Multiplex nucleic acid amplification

ABSTRACT

In some embodiments, the disclosure relates generally to compositions, comprising a single reaction mixture containing a plurality of different populations of discrete supports, and a plurality of different populations of target nucleic acids. The single reaction mixture can contain a first population of beads; a second population of beads; a first population of target nucleic acids, where at least two different target nucleic acids in the first population of target nucleic acids can bind to a bead in the first population of beads; and a second population of target nucleic acids, where at least two different target nucleic acids in the second population of target nucleic acids can bind to a bead in the second population of beads. The single reaction mixture can be employed to monoclonally amplify the first target nucleic acids on the first beads, and monoclonally amplify the second target nucleic acids on the second beads.

This application claims priority to U.S. Provisional Application No.62/012,213, filed Jun. 13, 2014, and to U.S. Provisional Application No.62/113,257, filed Feb. 6, 2015, each of which are incorporated byreference in its entirety herein.

Throughout this application various publications, patents, and/or patentapplications are referenced. The disclosures of the publications,patents and/or patent applications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Many nucleic acid analysis platforms, including “next-gen sequencing”(NGS), PCR and/or genetic analysis platforms, utilize beads or singlesurfaces (e.g., arrays) that are attached with one or more monoclonalamplicons (e.g., templated beads or templated arrays). These templatedbeads and/or arrays are typically generated via amplification-basedmethods. For example, templated beads can be formed using methods suchas emulsion PCR, which employs water-in-oil emulsions. This type ofemulsion forms numerous aqueous micro-droplets that are partitioned fromeach other by an oil phase. Ideally, the micro-droplets contain a singletemplate nucleic acid and one bead, along with enzymes, nucleotides andother reagents for conducting a nucleic acid amplification reaction.Each micro-droplet serves as a separate compartment within which clonalamplification occurs. When such micro-droplets are incubated undersuitable amplification conditions, the individual micro-droplets yieldone bead attached to monoclonal amplicons of the template nucleic acid.In other methods, nucleic acid templates can be mixed with beads andamplified clonally onto beads without need for emulsions. See, e.g.,U.S. Ser. No. 13/328,844 (U.S. Patent Publication No. 20120156728) andSer. No. 13/842,296 (U.S. Patent Publication No. 20130225421),incorporated by reference herein in their entireties. However, suchmethods typically require tightly controlled dilution and/or carefuladjustment of the relative concentrations of the template nucleic acidsand beads in order to dilute the number of nucleic acid molecules suchthat only one template nucleic acid molecule is amplified onto any givenbead. This can be challenging to achieve. For example, at least some ofthe micro-droplets in an emulsion will often contain multiple differenttemplate nucleic acids, which leads to formation of polyclonal beadsthat might not yield useful information in a downstream assay. Othermicro-droplets may contain one template nucleic acid molecule andmultiple beads, which leads to multiple beads each attached tomonoclonal amplicons of the same template nucleic acid, which results induplicate sequencing reads. Polyclonality and duplicate reads aresignificant problems for interpretation of next gen sequencing data.There is a need for improved nucleic acid amplification methods forclonal amplification of multiple templates in parallel with higher yieldand throughput. There is also a need for clonal amplification methodsthat avoid the need for limiting dilution, and methods that allow use ofhigher effective concentrations of templates and primers within theamplification reaction mixture while sufficiently preserving clonality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic that depicts one embodiment of the compositions,as well as related, systems, methods, kits and apparatuses for nucleicacid synthesis. One bead from a first plurality of first types of beads(100) attached with a first capture primer (110), one target nucleicacid from a first population of target nucleic acids (140) that includesa first adaptor (120) and a second adaptor (130), and a first reverseprimer (150). FIG. 1 also depicts one bead from a second plurality ofsecond types of beads (200) attached with a second capture primer (210),one target nucleic acid from a second population of target nucleic acids(240) that includes a third adaptor (220) and a fourth adaptor (230),and a second reverse primer (250). The first and second beads, the firstand second target nucleic acids, and the first and second reverseprimers can be contained in a single reaction mixture.

FIG. 2 is a schematic that depicts one embodiment of the compositions,as well as related, systems, methods, kits and apparatuses for nucleicacid synthesis. One bead from a first plurality of first types of beads(100) attached with a first capture primer (110), one target nucleicacid from a first population of target nucleic acids (140) that includesa first adaptor (120) and a second adaptor (130), a first reverse primer(150), and a first fusion primer (160). FIG. 2 also depicts one beadfrom a second plurality of second types of beads (200) attached with asecond capture primer (210), one target nucleic acid from a secondpopulation of target nucleic acids (240) that includes a third adaptor(220) and a fourth adaptor (230), a second reverse primer (250), and asecond fusion primer (260). The first and second beads, the first andsecond target nucleic acids, the first and second reverse primers, andthe first and second fusion primers can be contained in a singlereaction mixture.

FIG. 3A is a schematic that depicts one embodiment of the compositions,as well as related, systems, methods, kits and apparatuses for nucleicacid synthesis. One target nucleic acid from a first population oftarget nucleic acids (140) that includes a first adaptor (120) and asecond adaptor (130), and a first conversion primer (170 a/b) which canbe a fusion primer. Optionally, the first conversion primer (170 a/b)includes a sequence (170 a) that is not contained in, or is notcomplementary to, a portion of the first adaptor (120). In one exemplaryembodiment, the first conversion primer (170 a/b) can be used in aprimer extension reaction to append a conversion adaptor (170 a) to thefirst adaptor (120) to yield a first nucleic acid molecule havingsequences 170 a, 120, 140 and 130. FIG. 3A also depicts one targetnucleic acid from a second population of target nucleic acids (240) thatincludes a third adaptor (220) and a fourth adaptor (230), and a secondconversion primer (270 a/b) which can be a fusion primer. Optionally,the second conversion primer (270 a/b) includes a sequence (270 a) thatis not contained in, or is not complementary to, a portion of the thirdadaptor (220). In another exemplary embodiment, the second conversionprimer (270 a/b) can be used in a primer extension reaction to append aconversion adaptor sequence (270 a) to the third adaptor (220) to yielda second nucleic acid molecule having sequences 270 a, 220, 240 and 230.The first and second target nucleic acids, and the first and secondconversion primers can be contained in a single reaction mixture.

FIG. 3B is a schematic that depicts one embodiment of the compositions,as well as related, systems, methods, kits and apparatuses for nucleicacid synthesis. One bead from a first plurality of first types of beads(100) attached with a first capture primer (110), one target nucleicacid from a first population of target nucleic acids (140) that includesa first conversion adaptor (170 a) and a first adaptor (120) and asecond adaptor (130), a first reverse primer (150), and a first fusionprimer (160). FIG. 3B also depicts one bead from a second plurality ofsecond types of beads (200) attached with a second capture primer (210),one target nucleic acid from a second population of target nucleic acids(240) that includes second conversion adaptor (270 a) and a thirdadaptor (220) and a fourth adaptor (230), a second reverse primer (250),and a second fusion primer (260). The first and second beads, the firstand second target nucleic acids, the first and second reverse primers,and the first and second fusion primers can be contained in a singlereaction mixture.

FIG. 4A shows a fluorescence profile of a first batch of templated beadsprepared using four different beads attached with capture primers andtheir cognate adaptors, fusion primers, and reverse primers.

FIG. 4B shows a fluorescence profile of a second batch of templatedbeads prepared using four different beads attached with capture primersand their cognate adaptors, fusion primers, and reverse primers.

FIG. 5A shows a fluorescence profile of a first batch of templated beads(pre-enriched) prepared using four different beads attached with captureprimers and their cognate adaptors, fusion primers, and reverse primers.

FIG. 5B shows a fluorescence profile of the same first batch oftemplated beads shown in FIG. 5A (enriched) prepared using fourdifferent beads attached with capture primers and their cognateadaptors, fusion primers, and reverse primers.

FIG. 5C shows a fluorescence profile of a second batch of templatedbeads (pre-enriched) prepared using four different beads attached withcapture primers and their cognate adaptors, fusion primers, and reverseprimers.

FIG. 5D shows a fluorescence profile of the same first batch oftemplated beads shown in FIG. 5C (enriched) prepared using fourdifferent beads attached with capture primers and their cognateadaptors, fusion primers, and reverse primers.

FIG. 6 is a graph showing duplicate rates of separate batches oftemplated beads using one, two, three or four different types of beadsattached with capture primers and their cognate adaptors, fusionprimers, and reverse primers.

FIG. 7 is a graph showing duplicate rates of separate batches oftemplated beads using one, two, three or four different types of beadsattached with capture primers and their cognate adaptors, fusionprimers, and reverse primers. The resulting templated beads were loadedonto one, three, or four Ion Torrent™ Proton I™ chips, and sequenced.

DETAILED DESCRIPTION

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, foramplifying different template nucleic acids with different discretesupports (e.g., beads), or onto different discrete regions of a singlesupport (e.g., arrays). The advantages provided by the methods, as wellas related, systems, compositions, kits and apparatuses includegenerating fewer polyclonal amplicons while producing more monoclonalamplicons (or at least substantially monoclonal amplicons). Theadvantages also include reducing duplicate sequencing reads.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, foramplifying different target nucleic acids by one or more rounds ofnucleic acid synthesis. In some embodiments, the methods can includeamplifying multiple different target nucleic acids onto beads, toproduce a plurality of templated beads, where each bead is attached to asubstantially monoclonal population of one of the target nucleic acids.In some embodiments, the methods, as well as related, systems,compositions, kits and apparatuses, comprise amplifying the differenttarget nucleic acids with multiple beads, in parallel, in a singlereaction mixture. In some embodiments, the single reaction mixture canbe compartmentalized (e.g., via emulsion), or alternatively the singlereaction mixture can lack compartmentalization. In some embodiments, themethods include reducing the production of polyclonal amplicons withinthe reaction mixture. The reducing optionally includes increasing theaverage distance between the individual target nucleic acids to beamplified within the single reaction mixture. In some embodiments,reducing the production of polyclonal amplicons can include increasingthe volume of the reaction mixture. It is understood that applyingsuitable techniques, such as those described herein, can result in bothincreasing the volume of the reaction mixture and increasing the averagedistance between individual target nucleic acids to be amplified.

In some embodiments, the average distance between target nucleic acidswithin the reaction mixture and/or the volume of the reaction mixturecan be increased by adding additional components to the reactionmixture. In some embodiments, the additional components can include oneor more discrete physical components, such as particles, beads,supports, and the like. In some embodiments, the discrete physicalcomponents can be useful in maintaining the separation between twodifferent target nucleic acids to be amplified. In some embodiments, thediscrete physical components can be useful in increasing the effectivedistance between two target nucleic acids to be amplified. “Effectivedistance”, as used herein, refers to the average path length taken by afirst target nucleic acid to travel from its current location to thelocation of a second target nucleic acid within the same reactionmixture.

In some embodiments, the additional components in the reaction mixturecan bind to migrating or diffusible target nucleic acids. In someembodiments, the additional components can bind non-specifically tomigrating or diffusible target nucleic acids. In some embodiments, theadditional components can bind to one or more specific nucleotidesequences on at least a portion of the migrating or diffusible targetnucleic acids. In some embodiments, the sequence-specificity of thenucleotide sequence binding of the additional components can be providedby one or more capture primers. The capture primers are optionallyattached to one or more beads, or to one or more locations on a surface.In some embodiments, the capture primers are attached to at least one ofthe additional components in the reaction mixture. In some embodiments,the capture primers include sequences that are complementary to at leasta portion of the target nucleic acids. In some embodiments, the captureprimers attached to or otherwise associated with the beads, surfaceand/or additional components are random or degenerate capture primers,such that the random or degenerate primers are able to bind a pluralityof complementary sequence portions on migrating or diffusible targetnucleic acids. In some embodiments, the capture primers attached to thebeads, surface and/or additional components are capable oftemplate-directed elongation. For example, the capture primersoptionally have a terminal 3′ OH group. In some embodiments, the captureprimers include an extendible 3′ terminal end. In some embodiments, thecapture primers attached to the additional components are incapable oftemplate-directed elongation, such as by modification of the 3′-hydroxylgroup on the 3′ terminal nucleotide of the oligonucleotides. In thismanner, the extension-incapable primer can bind to migrating ordiffusible target nucleic acid, either in a sequence-specific orsequence-non-specific manner, but because they are not capable offurther template-directed elongation they will not contribute to furtheramplification that may lead to polyclonality. In some embodiments, the3′ terminal nucleotide of a capture primer is a dideoxynucleotide, suchthat the capture primer is elongation-incompetent. In some embodimentsthese elongation-incompetent capture primers are modified, such as bymodified internucleotide linkages (e.g., thiophosphate) or blockinggroups, so as to be resistant to exonucleases or proof-reading, such as3′-5′ exonuclease activity. In some embodiments, one or more captureprimers can be attached to a particle, bead, or support, where thecapture primer binds selectively, or non-selectively, to a targetnucleic acid, and where the capture primer is capable of supporting aprimer extension reaction.

Without intending to be bound by any particular model or theory, byadding the additional components (e.g., beads, particles or supports)described above to the amplification reaction mixture, the additionalcomponents serve to surround and separate the target nucleic acids fromeach other, thereby increasing the effective distance between theindividual target nucleic acid molecules, and thus increasing the actualdistance that migrating target nucleic acids must traverse to encounteranother template location, which may result in polyclonality. In someembodiments, the presence of the additional components may alsophysically impede or block the migration of target nucleic acids withinthe reaction mixture, since the additional components are typicallyimpermeable to migrating target nucleic acids. In some embodiments, theadditional components are essentially impermeable to the migratingtarget nucleic acids as the components are of a different phase ofmatter (e.g. solid or gas) than the reaction mixture (e.g. liquid).

In some embodiments, the addition of the additional components, such asbeads, capture primers and/or adaptors to the reaction mixture can serveto reduce or eliminate target nucleic acid migration and polyclonalityin small volumes of reaction mixtures. In particular, reducing thevolume of the amplification reaction mixture may be desirable in certainembodiments as it can improve efficiency of the reaction, or reduce theamount of needed reagents, or reduce the size of the reaction vessel, orother advantages or any combination thereof. However, reducing thevolume may also decrease the distance between different templatemolecules in the reaction mixture, thereby potentially increasing theproblem of target nucleic acid migration and hence polyclonality. Thus,in some embodiments, by also introducing the additional components asdescribed herein, the migration of target nucleic acids that may causepolyclonality can be reduced, mitigated or prevented despite the smallreaction volume as described herein.

In some embodiments, the addition of additional components describedabove to the reaction mixture, which contains the different targetnucleic acids to be amplified, serves to increase the total volume ofthe reaction volume. In such embodiments, and without intending to bebound by any model or theory, increasing the volume may increase themean distance between any two or more different target nucleic acids inthe reaction mixture. In this manner, by increasing the mean distance,the likelihood of migrating target molecules traversing the distancebetween different target molecules, and thus reduce, mitigate or preventpolyclonality from amplification of different target nucleic acids at agiven amplification location.

The disclosed methods (and related compositions, systems, kits andreagents) can offer several advantages as compared to conventionalamplification methods, including emPCR. For example, conventional PCRworkflows are time-consuming, and waste equipment and reagents ascompared to the disclosed methods. For example, to conduct multiplexsequencing on a high throughput system, separate batches of templatedbeads must be prepared (each batch being different from the otherbatches), which requires setting-up multiple amplification reactionmixtures in separate reaction vessels, each vessel containing differentbeads and/or different target nucleic acids and/or separate reagents.The resulting separate batches of different types of templated beads arepooled together to make a mixture. The throughput of each separateamplification reaction is typically limited by Poisson statistics. Forexample, the proportion of productive droplets in an emPCR reaction(including the proportion of droplets yielding monoclonal orsubstantially monoclonal amplified populations, or the proportion ofdroplets yielding monoclonal or substantially monoclonal templatedbeads) is typically limited according to Poisson statistics to thenumber of droplets that receive a single template nucleic acid.

In contrast, the nucleic acid synthesis methods, according to thepresent teachings, provide advantages not offered by the conventionalamplification methods. The nucleic acid synthesis methods yield asimplified workflow, from the bead templating step to the sequencingstep, because a mixture of different types of templated beads isprepared in a single reaction vessel, using a single reaction mixture(with or without emulsions), with (i) different bead types, each beadtype attached with different capture primers, and with (ii) differentpopulations of target nucleic acids, each population having differentadaptor sequences joined to the target nucleic acids, where thedifferent adaptor sequences bind selectively to one of the differenttypes of capture primers. Thus, the simplified methods do not requiremultiple reaction vessels, use reduced amounts of reagents, and do notemploy a pooling step.

Many next generation sequencing workflows involve at least one nucleicacid amplification step. The amplification step can introduceamplification biases, which include polyclonality and duplication. Thesebiases are problematic because they distort the original complexity(e.g., relative abundance) of the genetic material to be sequenced.

The disclosed methods, as well as related, systems, compositions, kitsand apparatuses, offer advantages over standard emulsion PCR procedures,by reducing both polyclonality and duplication rates of the templatedbeads.

The disclosed methods also provide increased throughput and reactionefficiency by overcoming Poisson-based limitations based on therequirement to compartmentalize or otherwise isolate single templatenucleic acid molecules to achieve clonal amplification. For example, thedisclosed methods overcome Poisson-based constraints and allow theamplification of different types of templates onto different types ofbeads within a single amplification reaction, optionally within a singledroplet within an emulsion-based amplification reaction.

The standard emPCR methods require meticulous dilution and adjustment ofthe relative concentrations of the beads and target nucleic acids sothat, ideally each aqueous droplet in the emulsion receives no more thana single target nucleic acid, and a single bead that is attached withcapture primers. For example, due to Poisson-based constraints,typically about 20-35% of the templated beads generated by standardemulsion PCR procedures are polyclonal. In a massively parallelsequencing system, the monoclonal and polyclonal templated beads areloaded together on the same sequencing apparatus (e.g., wells, grooves,and flowcells). The polyclonal beads do not yield meaningful sequencinginformation, because each bead is templated with different targetsequences. Thus, the presence of polyclonal beads on the sequencingapparatus reduces the overall percent of useful sequencing reads, andreduces sequencing throughput. In contrast, templated beads preparedaccording to the present teachings can have reduced polyclonality ratesof about 12-30%, which produce improved overall useful sequencinginformation that leads to an increase in the number of sequencing readsobtained from a single sequencing run, and increased total sequencingthroughput. The present teachings provide methods for improvingpolyclonality rates without dilution and/or adjustment of the relativeconcentrations of the beads and target nucleic acids, and are conducteda single reaction vessel that contains a single reaction mixture,optionally with an emulsion

An increase in the number of sequencing reads can also be achieved byproducing enough templated beads to load the greatest number ofavailable sites (e.g., wells, grooves, flowcell, and the like) on thesequencing apparatus. The standard emPCR procedures do not produceenough templated beads, therefore multiple reactions must be prepared,and the templated beads are pooled and loaded onto the sequencingapparatus.

The nucleic acid synthesis methods, according to the present teachings,can be used to prepare emulsions with an increase in the number ofaqueous droplets, without a change in reaction volume, to yield anincrease in the number of templated beads. This will obviate the need toprepare multiple amplification reactions and bead pooling, and willincrease the number of sequencing reads.

When conducting a standard emPCR procedure, changes in reaction volumewill also alter the yield of duplicate beads. For example, a decrease inthe amplification reaction volume will increase the duplicate bead rate.

The nucleic acid synthesis methods, according to the present teachings,can be conducted in a reduced total reaction volume withoutsignificantly increasing the yield of duplicate beads. For example,using the nucleic acid synthesis method (with emulsion), templated beadswere prepared in about 2.4 mL reaction volume and yielded about 3.8%duplicate beads, compared to templated beads prepared in about 1.2 mLreaction volume that yielded about 9.4% duplicate beads. In contrast, acontrol emPCR reaction was conducted in about 1.2 mL and yielded about26% duplicate beads. The nucleic acid synthesis methods, according tothe present teachings, can be conducted in reaction volumes as small as600 μL or smaller volumes.

A standard emPCR reaction will produce templated beads having about15-20% duplicate beads. Duplicate beads in a massively parallelsequencing system are undesirable for analyses that compare abundances.For example, a starting pool of RNA may contain different abundances ofdifferent transcript species. The relative abundances of the differentRNA species will be misrepresented by templated bead preparations thathave high duplicate rates.

Templated beads prepared by the nucleic acid synthesis methods,according to the present teachings, have a significantly reducedduplicate bead rate of about 2-12% (using RNA or DNA as startingmaterial), which makes interpretation of the sequencing data moreclosely aligned with the complexity of the original genetic material.

Another example of amplification bias includes batch effect, whicharises from variations in conducting different reactions on the same dayor on different days, for example, due to pipetting inaccuracies,reagent batch differences, reaction conditions, and differences in thetechnicians who are conducting the amplification reactions. Anothersource of batch effect comes from different primer and/or adaptorsequences that exhibit slight differences in amplification efficiencies.Under standard emPCR conditions, the different amplificationefficiencies can be exacerbated when amplified in separate reactionvessels (e.g., separate batches).

The nucleic acid synthesis methods, according to the present teachings,can be used to prepare a mixture of different types of templated beadswith reduced batch bias, by conducting the amplification reaction in asingle reaction vessel, using a single reaction mixture (with or withoutemulsions).

The nucleic acid synthesis methods, according to the present teachings,can be used to reduce bead clumping that may occur in a single reactionnucleic acid synthesis method. For example, any of the capture primers,adaptors, reverse primers, fusion primers, and/or conversion primers cancause bead clumping, which may lead to producing polyclonal beads orduplicate beads. Without wishing to be bound by theory, we postulatethat bead clumping may be caused by primer dimer interaction between twoor more different types of capture primers attached to beads. Oneapproach to solving the bead clumping problem includes selecting thelength and/or sequence of any of the primers to reduce bead clumping,where the primers include any one or any combination of the captureprimers, adaptors, reverse primers, fusion primers, and/or conversionprimers.

The nucleic acid synthesis methods, according to the present teachings,can be used to reduce duplicate bead formation that may occur in asingle reaction nucleic acid synthesis method. For example, any of thecapture primers, adaptors, reverse primers, fusion primers, and/orconversion primers can cause duplicate bead formation. Without wishingto be bound by theory, we postulate that bead clumping may lead toincreased production of duplicate template beads. One approach tosolving the duplicate bead formation problem includes selecting thelength and/or sequence of any of the primers to duplicate beadformation, where the primers include any one or any combination thecapture primers, adaptors, reverse primers, fusion primers, and/orconversion primers. Another approach includes increasing the number ofdifferent types of beads in the nucleic acid synthesis method. Forexample, duplicate bead formation can be reduced by increasing thenumber of different types beads used to conduct the nucleic acidsynthesis method from two types, to three, four, five, six, or moredifferent types of beads. In some embodiments, reduced duplicate beadformation yields improved sequencing metrics, including aligned reads,coverage, polyclonality and mean read length.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprising aplurality of different populations of supports, and a plurality ofdifferent populations of target nucleic acids. In some embodiments, thedifferent populations of target nucleic acids can each bind (e.g., bindselectively) to a correspond population of beads. In some embodiments,the compositions (and related methods, systems, kits and apparatuses)also includes any one or any combination of: primers (e.g., captureprimer, fusion primer, and/or reverse primers), enzymes (e.g.,polymerases), accessory proteins (e.g., recombinase, recombinase loadingprotein, single-stranded binding protein, helicase or topoisomerase),nucleotides, divalent cations, binding partners and/or co-factors.

In some embodiments, the plurality of different populations of discretesupports, and the plurality of different populations of target nucleicacids, are contained in a single reaction mixture. In some embodiments,the single reaction mixture comprises a single amplification reactionmixture. In some embodiments, the plurality of different populations ofdiscrete supports, and a plurality of different populations of targetnucleic acids, can be contained in a single reaction vessel.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least two different populations of discrete supports (e.g., beads andthe like) and at least two different populations of target nucleicacids. In some embodiments, the at least two different population ofdiscrete supports are each attached to a particular nucleic acidsequence, e.g., a capture primer sequence. In some embodiments, eachdifferent population of supports is attached to a different captureprimer sequence. In some embodiments, the discrete supports are eachattached to one or more capture primers that provide binding, viahybridization, to their corresponding population of target nucleic acidsthat can hybridize to the capture primers. In some embodiments, thediscrete supports comprise a plurality of particles or beads. In someembodiments, the compositions (and related methods, systems, kits andapparatuses) also includes any one or any combination of: additionalprimers (e.g., capture primer, fusion primer, and/or reverse primers),enzymes (e.g., polymerases), accessory proteins (recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase), nucleotides, divalent cations, binding partnersand/or co-factors. In some embodiments, at least two differentpopulations of discrete supports are each bound to a differentpopulation of target nucleic acids in a single reaction mixture. In someembodiments, the at least two different populations of discrete supportsand at least two different populations of target nucleic acids can becontained in a single amplification reaction mixture. In someembodiments, the at least two different populations of discrete supportsand at least two different populations of target nucleic acids can becontained in a single reaction vessel. In some embodiments, thecompositions (and related methods, systems, kits and apparatuses)comprise more than two different populations of discrete supports andmore than two different populations of target nucleic acids.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising (i) atleast a first population of beads, (ii) at least a first population oftarget nucleic acids, (iii) at least a second population of beads, and(iv) at least a second population of target nucleic acids.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) comprise a single reaction mixture that includes(i) at least a first population of beads, (ii) at least a firstpopulation of target nucleic acids, (iii) at least a second populationof beads, and (iv) at least a second population of target nucleic acids.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture that also includes any one or any combination of:additional primers (e.g., capture primer, fusion primer, and/or reverseprimers), enzymes (e.g., polymerases), accessory proteins (recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase), one or more nucleotides, divalent cations, bindingpartners and/or co-factors.

In some embodiments, the first population of beads can bind the firstpopulation of target nucleic acids (FIG. 1). Optionally, the bindingbetween the first population of beads and the first population of targetnucleic acids includes nucleic acid hybridization. Optionally the beadsof the first population include first capture primers and the bindingincludes hybridizing the target nucleic acids of the first population tothe first capture primers. Optionally, the beads of the first populationinclude first capture primers and no other types of capture primers.

In some embodiments, the first population of beads is bound to the firstpopulation of target nucleic acids.

In some embodiments, one or more first type of capture primers areattached to the first population of beads.

In some embodiments, the first population of target nucleic acidscontains a plurality of target nucleic acids having the same sequencesand/or a plurality of target nucleic acids having different sequences.In some embodiments, the target nucleic acids of the first populationinclude a common sequence (“first common sequence”) that is present insome or all members of the first population. In some embodiments, thetarget nucleic acids of the first population each include one or morecommon sequences and optionally one or more additional sequences thatare not common. In some embodiments, the first common sequence caninclude a primer binding site (“first primer binding site”) that iscomplementary or identical to a first capture primer.

In some embodiments, the first population of beads is attached to one ormore first capture primers that can hybridize to the first population oftarget nucleic acids or their complements. In some embodiments, thefirst capture primers can hybridize to the first primer binding site orits complement.

In some embodiments, the second population of beads can bind the secondpopulation of target nucleic acids (FIG. 1). Optionally, the bindingbetween the second population of beads and the second population oftarget nucleic acids includes nucleic acid hybridization. Optionally thebeads of the second population include second capture primers and thebinding includes hybridizing the target nucleic acids of the secondpopulation to the second capture primers. Optionally, the beads of thesecond population include second capture primers and no other types ofcapture primers.

In some embodiments, the second population of beads is bound to thesecond population of target nucleic acids. Optionally, the bindingbetween the second population of beads and the second population oftarget nucleic acids includes nucleic acid hybridization.

In some embodiments, one or more second type of capture primers areattached to the second population of beads.

In some embodiments, the second population of target nucleic acidscontains a plurality of target nucleic acids having the same sequencesand/or a plurality of target nucleic acids having different sequences.In some embodiments, the target nucleic acids of the second populationinclude a common sequence (“second common sequence”) that is present insome or all members of the second population. In some embodiments, thetarget nucleic acids of the second population each include one or morecommon sequences and optionally one or more additional sequences thatare not common. In some embodiments, the second common sequence caninclude a primer binding site (“second primer binding site”) that iscomplementary or identical to a second capture primer.

In some embodiments, the second population of beads is attached to oneor more second capture primers that can hybridize to the secondpopulation of target nucleic acids or their complements. In someembodiments, the second capture primers can hybridize to the secondprimer binding site or its complement.

In some embodiments, the at least a first population of beads, the atleast a first population of target nucleic acids, the at least a secondpopulation of beads, and the at least a second population of targetnucleic acids, are contained in a single reaction mixture.

The single reaction mixture can further include a third population oftarget nucleic acids and a third population of beads. The thirdpopulation of target nucleic acids can include a third common sequencecontaining a third primer binding site. The third population of beadscan include a third capture primer that is complementary or identical tothe third primer binding site.

The single reaction mixture can further include a fourth population oftarget nucleic acids and a fourth population of beads. The fourthpopulation of target nucleic acids can include a fourth common sequencecontaining a fourth primer binding site. The fourth population of beadscan include a fourth capture primer that is complementary or identicalto the fourth primer binding site.

The single reaction mixture can further include a fifth, sixth, seventh,eighth, ninth, tenth or higher order population of target nucleic acidsand a fifth, sixth, seventh, eighth, ninth, tenth or higher orderpopulation of beads. The fifth, sixth, seventh, eighth, ninth, tenth orhigher order population of target nucleic acids can include a fifth,sixth, seventh, eighth, ninth, tenth or higher order common sequencecontaining a fifth, sixth, seventh, eighth, ninth, tenth or higher orderprimer binding site. The fifth, sixth, seventh, eighth, ninth, tenth orhigher order population of beads can include a fifth, sixth, seventh,eighth, ninth, tenth or higher order capture primer that iscomplementary or identical to the fifth, sixth, seventh, eighth, ninth,tenth or higher order primer binding site.

In some embodiments, the single reaction mixture comprises a singleamplification reaction mixture.

In some embodiments, the at least a first population of beads, the atleast a first population of target nucleic acids, the at least a secondpopulation of beads, and the at least a second population of targetnucleic acids, are contained in a single reaction vessel.

Single reaction mixture also includes any one or any combination of:additional primers (e.g., capture primer, fusion primer, and/or reverseprimers), enzymes (e.g., polymerases and/or recombinases), accessoryproteins, one or more nucleotides, divalent cations, affinity moietiesand/or co-factors.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture that contains (i) a first population of beads, thebeads of the first population being attached to one or more firstcapture primers; (ii) a second population of beads, the beads of thesecond population being attached to one or more second capture primers,wherein the first and second capture primers are different; (iii) afirst population of target nucleic acids, wherein the first populationincludes at least two different target nucleic acids that can each bindindependently to the first capture primers; and (iv) a second populationof target nucleic acids, wherein the second population includes at leasttwo different target nucleic acids that can each bind independently tothe second capture primers. Optionally, the first population of targetnucleic acids includes a primer binding sequence for binding the firstcapture primers. Optionally, the second population of target nucleicacids includes a primer binding sequence for binding the second captureprimers. Optionally, the single reaction mixture includes any one or anycombination of: additional primers (e.g., capture primer, fusion primer,and/or reverse primers), enzymes (e.g., polymerases), accessory proteins(recombinase, recombinase loading protein, singe-stranded bindingprotein, helicase or topoisomerase), one or more nucleotides, divalentcations, binding partners and/or co-factors.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising (i) afirst plurality of beads of a first type, (ii) a second plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids. In someembodiments, the beads of the first type include first capture primers.In some embodiments, the beads of the second type include second captureprimers. The first capture primers can be different from the secondcapture primers. In some embodiments, the first population of targetnucleic acids includes a first primer binding sequence that can bind tothe first capture primers. The second population of target nucleic acidscan include a second primer binding sequence that can bind to the secondcapture primers. In some embodiments, a composition according to thedisclosure is present within a single reaction mixture. In someembodiments, the composition includes a polymerase and/or nucleotidesand/or other amplification reagents. In some embodiments, thecomposition is present within an emulsion microdroplet. In someembodiments, the composition further includes beads of a third typeattached to third capture primers. The composition can further include athird population of target nucleic acids including third primer bindingsequences, wherein the third primer binding sequence can hybridize tothe third capture primers but not to the first and second captureprimers.

In some embodiments, the composition further includes beads of a fourthtype attached to fourth capture primers. The composition can furtherinclude a fourth population of target nucleic acids including fourthprimer binding sequences, wherein the fourth primer binding sequence canhybridize to the fourth capture primers but not to the first, second andthird capture primers.

In some embodiments, the composition further includes beads of a fifthtype attached to fifth capture primers. The composition can furtherinclude a fifth population of target nucleic acids including fifthprimer binding sequences, wherein the fifth primer binding sequence canhybridize to the fifth capture primers but not to the first, second,third and fourth capture primers.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture which (i) a first plurality of beads of a first type,(ii) a second plurality of beads of a second type, (iii) a firstpopulation of target nucleic acids, and (iv) a second population oftarget nucleic acids.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) comprise a single reaction mixture which (i) afirst plurality of beads of a first type, (ii) a second plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids, where thesingle reaction mixtures does not provide compartmentalization orpartitioning.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) comprise a single reaction mixture that contains(i) a first plurality of beads of a first type, the beads of the firsttype being attached to one or more first capture primers; (ii) aplurality of beads of a second type, the beads of the second type beingattached to one or more second capture primers, wherein the first andsecond capture primers are different; (iii) a first population of targetnucleic acids, wherein the first population includes at least twodifferent target nucleic acids that can each bind independently to afirst capture primer; and (iv) a second population of target nucleicacids, wherein the second population includes at least two differenttarget nucleic acids that can each bind independently to a secondcapture primer. In some embodiments, the single reaction mixture doesnot provide compartmentalization.

In some embodiments, the beads of the first type can be attached to oneor more first capture primers.

In some embodiments, the beads of the second type can be attached to oneor more second capture primers.

In some embodiments, the first population of target nucleic acidsincludes at least two different target nucleic acids that can each bindindependently to a first capture primer.

Optionally, at least two different target nucleic acids in the firstpopulation of target nucleic acids include a primer binding sequence(“first primer binding sequence”) that can hybridize to the firstcapture primer or its complement.

In some embodiments, the second population of target nucleic acidsincludes at least two different target nucleic acids that can each bindindependently to a second capture primer.

Optionally, at least two different target nucleic acids in the secondpopulation of target nucleic acids include a primer binding sequence(“second primer binding sequence”) that can hybridize to the secondcapture primer or its complement.

In some embodiments, the single reaction mixture comprises a singlecontinuous liquid phase. Optionally, the single continuous liquid phasecomprises an aqueous phase liquid. Optionally, the single continuousliquid phase lacks a hydrophobic phase.

In some embodiments, the single continuous liquid phase does notpartition the first type of beads from the second type of beads. In someembodiments, the single continuous liquid phase does not partition thefirst population of target nucleic acids from the second population oftarget nucleic acids. In some embodiments, the single continuous liquidphase does not partition any of the types of beads from any of the typesof target nucleic acids.

In some embodiments, a single reaction vessel contains the singlereaction mixture which includes the first and second plurality of beadsof the first and second type, respectively, and the first and secondpopulation of target nucleic acids, where the single reaction mixture isnot compartmentalized or partitioned.

In some embodiments, the first population of target nucleic acidscontains target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the first population of target nucleic acids contains two ormore target nucleic acids that can bind the first plurality of beads.

Optionally, the binding between the first plurality of beads and thefirst population of target nucleic acids comprise selective binding thatincludes Watson-Crick base pairing between two nucleic acids.

Optionally, one or more first type of capture primers are attached tothe first plurality of beads.

Optionally, the first population of target nucleic acids contains two ormore different target nucleic acids that can bind independently to firstcapture primers.

Optionally, first plurality of beads is attached with one or more of afirst type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the first type of capture primers are attached covalently tothe first plurality of beads.

Optionally, the first plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the first plurality of beads do not include any secondcapture primers.

Optionally, the first type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the first population of targetnucleic acids are bound to the first plurality of beads.

Optionally, the target nucleic acids of the first population of targetnucleic acids are bound to the first plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the firstpopulation of target nucleic acids are each hybridized to a firstcapture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has a sequence that is complementaryor identical to at least a region of a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

In some embodiments, the second population of target nucleic acidscontains target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the second population of target nucleic acids contains twoor more target nucleic acids that can bind the second plurality ofbeads.

Optionally, the binding between the second plurality of beads and thesecond population of target nucleic acids comprise selective binding.

Optionally, one or more second type of capture primers are attached tothe second plurality of beads.

Optionally, the second population of target nucleic acids contains twoor more different target nucleic acids that can bind independently tosecond capture primers.

Optionally, second plurality of beads is attached with one or more of asecond type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the second type of capture primers are attached covalentlyto the second plurality of beads.

Optionally, the second plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the second plurality of beads do not include any firstcapture primers.

Optionally, the second type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the second population of targetnucleic acids are bound to the second plurality of beads.

Optionally, the target nucleic acids of the second population of targetnucleic acids are bound to the second plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the secondpopulation of target nucleic acids are each hybridized to a secondcapture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has a sequence that is complementaryor identical to at least a region of a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the target nucleic acids in the first population of targetnucleic acids can have the same sequence or a mixture of differentsequences.

Optionally, the target nucleic acids in the second population of targetnucleic acids can have the same sequence or a mixture of differentsequences.

Optionally, at least one target nucleic acid from the first populationof target nucleic acids and at least one target nucleic acid from thesecond population of target nucleic have the same sequence.

Optionally, the first population of target nucleic acids and the secondpopulation of target nucleic acids do not contain any target nucleicacids with the same sequence.

Optionally, the first plurality of beads is attached with a first typeof capture primers, and the second plurality of beads is attached with asecond type of capture primers, and the sequences of the first type ofcapture primers and the second type of capture primers are the same ordiffer.

Optionally, the first plurality of beads includes only capture primersof the first type.

Optionally, the second plurality of beads includes only capture primersof the second type.

Optionally, the first population of beads binds selectively, viahybridization, to the first population of target nucleic acids.

Optionally, the second population of beads binds selectively, viahybridization, to the second population of target nucleic acids.

Optionally, the selective binding between the first population of beadsand the first population of target nucleic acids, and between the secondpopulation of beads and the second population of target nucleic acids,occurs in a single reaction mixture.

Optionally, the selective binding between the first population of beadsand the first population of target nucleic acids, and between the secondpopulation of beads and the second population of target nucleic acids,occurs in a single reaction mixture that provides nocompartmentalization.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) which comprise a single reaction mixture furtherincludes additional primer types, including third, fourth, fifth, ormore different primer types.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) which comprise a single reaction mixture furtherincludes any one or any combination of: additional primers (e.g.,capture primer, fusion primer, and/or reverse primers), enzymes (e.g.,polymerases), accessory proteins (recombinase, recombinase loadingprotein, single-stranded binding protein, helicase or topoisomerase),one or more nucleotides, divalent cations, binding partners and/orco-factors.

Optionally, the enzymes catalyze nucleotide incorporation (e.g.,polymerase).

Optionally, the enzymes comprise accessory proteins.

Optionally, the additional primers include primers in solution orattached to a support (e.g., a bead or particle).

Optionally, the nucleotides comprise natural nucleotides or analogsthereof.

Optionally, the binding partners comprise biotin.

Optionally, the co-factors include salts, cations, ATP, phosphocreatine,magnesium, manganese, and calcium.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction mixture.

Optionally, the single reaction mixture comprises an amplificationreaction mixture.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction vessel.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least two different types of beads and at least two different typesof target nucleic acids, in a single reaction mixture that providescompartmentalization or partitioning. In some embodiments, the singlereaction mixture provides at least one compartment that contains atleast two different types of beads and at least two different types oftarget nucleic acids. For example, the single reaction mixture has atleast one compartment that contains (1) a first plurality of beads of afirst type and a second plurality of beads of a second type, and (2) afirst population of target nucleic acids and a second population oftarget nucleic acids. Optionally, the at least one compartment cancontain additional different types of beads and additional differenttypes of target nucleic acids.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture which (i) a first plurality of beads of a first type,(ii) a second plurality of beads of a second type, (iii) a firstpopulation of target nucleic acids, and (iv) a second population oftarget nucleic acids, where the single reaction mixtures providescompartmentalization or partitioning. In some embodiments, the singlereaction mixture provides at least one compartment. Optionally, the atleast one compartment can contain additional different types of beadsand additional different types of target nucleic acids.

In some embodiments, the disclosure relates generally to thecompositions, and related methods, systems, kits and apparatuses,comprising a single reaction mixture that provides at least onecompartment, where the at least one compartment contains (i) a firstplurality of beads of a first type, the beads of the first type beingattached to one or more first capture primers; (ii) a plurality of beadsof a second type, the beads of the second type being attached to one ormore second capture primers, wherein the first and second captureprimers are different; (iii) a first population of target nucleic acids,wherein the first population includes at least two different targetnucleic acids that can each bind independently to a first captureprimer; and (iv) a second population of target nucleic acids, whereinthe second population includes at least two different target nucleicacids that can each bind independently to a second capture primer.

Optionally, the single reaction mixture is contained in a singlereaction vessel.

In some embodiments, the beads of the first type can be attached to oneor more first capture primers.

In some embodiments, the beads of the second type can be attached to oneor more second capture primers.

In some embodiments, the first population of target nucleic acidsincludes at least two different target nucleic acids that can each bindindependently to a first capture primer.

Optionally, at least two different target nucleic acids in the firstpopulation of target nucleic acids include a primer binding sequence forbinding the first capture primer.

In some embodiments, the second population of target nucleic acidsincludes at least two different target nucleic acids that can each bindindependently to a second capture primer.

Optionally, at least two different target nucleic acids in the secondpopulation of target nucleic acids include a primer binding sequence forbinding the second capture primer.

Optionally, the single reaction mixture further includes any one or anycombination of: additional primers (e.g., capture primer, fusion primer,and/or reverse primers), enzymes (e.g., polymerases), accessory proteins(recombinase, recombinase loading protein, single-stranded bindingprotein, helicase or topoisomerase), one or more nucleotides, divalentcations, binding partners and/or co-factors.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprising asingle reaction mixture that provides compartmentalization orpartitioning includes an emulsion.

Optionally, the emulsion comprises a discontinuous hydrophilic phase anda continuous hydrophobic phase.

Optionally, the discontinuous hydrophilic phase is surrounded by thecontinuous hydrophobic phase.

Optionally, the emulsion comprises at least one hydrophilic phasecompartment (e.g., droplet or micro-reactor) surrounded by a continuoushydrophobic phase.

Optionally, the discontinuous hydrophilic phase provides a compartment.

Optionally, the emulsion comprises a plurality of hydrophilic phasedroplets and a continuous hydrophobic phase.

Optionally, the emulsion comprises a plurality of aqueous droplets and acontinuous hydrophobic phase.

Optionally, the emulsion comprises at least one aqueous droplet.

Optionally, the at least one aqueous droplet includes one or more beads.

Optionally, the at least one aqueous droplet includes one or moredifferent target nucleic acids.

Optionally, the emulsion includes at least one aqueous droplet thatincludes one or more beads of the first type, the second type, or beadsof both the first and second type.

Optionally, the emulsion includes at least one aqueous droplet thatincludes one or more different target nucleic acids, including the firstpopulation, the second population, or both the first and secondpopulations of target nucleic acids.

In some embodiments, the emulsion comprises two immiscible liquidphases. In some embodiments, two immiscible liquid phases are mixedtogether to make the emulsion. In some embodiments, one of the liquidphases is dispersed in the other. Optionally, the emulsion comprises amixture of an aqueous liquid and a water-immiscible organic liquid.Optionally, the emulsion comprises at least one anionic, cationic ornon-ionic surfactant. Optionally, the emulsion can have a droplet-typedispersion comprising oil-in-water, water-in-oil, or a bicontinuousmicroemulsion.

In some embodiments, the water immiscible organic liquid comprises anoil. In some embodiments, the oil can be from a natural source,including animal (e.g., tallow or lard), fish (e.g., fish oil), shark,seeds, nuts or plants (e.g., vegetable oils). Optionally, the oil can befrom derived from petroleum, including mineral oils. Optionally, the oilcomprises a fluorochemical oil, polyalphaolefin or ester oil.

In some embodiments, the surfactant includes small molecule surfactants,polymeric surfactants, triblock co-polymer surfactants or non-ionicblock copolymer surfactants. Optionally, the surfactant comprises asorbitan oleate or a silicone surfactant.

Optionally, the hydrophilic phase compartment can contain at least twodifferent types of beads. Optionally, the hydrophilic phase compartmentcan contain at least two different types of target nucleic acids.

Optionally, at least one hydrophilic phase compartment can contain (i) afirst plurality of beads of a first type, (ii) a first population oftarget nucleic acids, (iii) a second plurality of beads of a secondtype, and (iv) a second population of target nucleic acids. Optionally,the at least one hydrophilic phase compartment can contain additionaldifferent types of beads and additional different types of targetnucleic acids. Optionally, the hydrophilic phase compartment can furthercontain any one or any combination of: primers (e.g., capture primer,fusion primer, and/or reverse primers), enzymes (e.g., polymerases),accessory proteins (recombinase, recombinase loading protein,single-stranded binding protein, helicase or topoisomerase), one or morenucleotides, divalent cations, binding partners and/or co-factors.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the first population of target nucleic acids whichinclude target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the first population of target nucleic acids contains two ormore target nucleic acids that can bind the first plurality of beads.

Optionally, the binding between the first plurality of beads and thefirst population of target nucleic acids comprise selective binding.

Optionally, the first plurality of beads are attached to one or morefirst type of capture primers.

Optionally, the first population of target nucleic acids contains two ormore different target nucleic acids that can bind independently to firstcapture primers.

Optionally, first plurality of beads is attached with one or more of afirst type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the first type of capture primers are attached covalently tothe first plurality of beads.

Optionally, the first plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the first plurality of beads do not include any secondcapture primers.

Optionally, the first type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the first population of targetnucleic acids are bound to the first plurality of beads.

Optionally, the target nucleic acids of the first population of targetnucleic acids are bound to the first plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the firstpopulation of target nucleic acids are each hybridized to a firstcapture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids is complementary to at least a regionof a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the second population of target nucleic acidswhich include target nucleic acids having the same sequence or a mixtureof different sequences.

Optionally, the second population of target nucleic acids contains twoor more target nucleic acids that can bind the second plurality ofbeads.

Optionally, the binding between the second plurality of beads and thesecond population of target nucleic acids comprise selective binding.

In some embodiments, one or more second type of capture primers areattached to the second plurality of beads.

Optionally, the second population of target nucleic acids contains twoor more different target nucleic acids that can bind independently tosecond capture primers.

Optionally, second plurality of beads is attached with one or more of asecond type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the second type of capture primers are attached covalentlyto the second plurality of beads.

Optionally, the second plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the second plurality of beads do not include any firstcapture primers.

Optionally, the second type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the second population of targetnucleic acids are bound to the second plurality of beads.

Optionally, the target nucleic acids of the second population of targetnucleic acids are bound to the second plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the secondpopulation of target nucleic acids are each hybridized to a secondcapture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids is complementary to at least a regionof a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains at least one target nucleic acid from the firstpopulation of target nucleic acids and at least one target nucleic acidfrom the second population of target nucleic with the same sequence.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the first population of target nucleic acids andthe second population of target nucleic acids which do not include anytarget nucleic acids with the same sequence.

Optionally, the first plurality of beads is attached with a first typeof capture primers, and the second plurality of beads is attached with asecond type of capture primers, and the sequences of the first type ofcapture primers and the second type of capture primers are the same ordifferent.

Optionally, the first plurality of beads includes only capture primersof the first type.

Optionally, the second plurality of beads includes only capture primersof the second type.

Optionally, the first population of beads binds selectively, viahybridization, to the first population of target nucleic acids.

Optionally, the second population of beads binds selectively, viahybridization, to the second population of target nucleic acids.

Optionally, the selective binding occurs in the at least one compartment(e.g., hydrophilic phase compartment).

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains additional primers, including third, fourth,fifth, or more different primer types.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture having at least one compartment (e.g., hydrophilicphase compartment) which contains (i) a first plurality of beads of afirst type, (ii) a first population of target nucleic acids, (iii) asecond plurality of beads of a second type, (iv) a second population oftarget nucleic acids, and (v) any one or any combination of: additionalprimers (e.g., capture primer, fusion primer, and/or reverse primers),enzymes (e.g., polymerases), accessory proteins (recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase), one or more nucleotides, divalent cations, bindingpartners and/or co-factors.

Optionally, the enzymes catalyze nucleotide incorporation (e.g.,polymerase).

Optionally, the enzymes comprise accessory proteins (e.g., recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase).

Optionally, the additional primers include primers in solution orattached to at least one discrete support. Optionally, the additionalprimers include primers that are not covalently attached to the first orsecond, or to any type of beads.

Optionally, the nucleotides comprise natural nucleotides or analogsthereof.

Optionally, the binding partners comprise biotin.

Optionally, the co-factors include salts, cations, ATP, phosphocreatine,magnesium, manganese, and calcium.

Optionally, the single reaction mixture comprises an amplificationreaction mixture.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction vessel.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprising areaction mixture containing a plurality of different template nucleicacids with a plurality of discrete supports (e.g., beads), and aplurality of target nucleic acid molecules (also referred to hereinsimply as “target nucleic acids”), wherein the beads and the targetnucleic acids are present in a specific amounts resulting in a definedratio of beads-to-target nucleic acids. In some embodiments, the ratioof beads-to-target nucleic acids is the number of beads present in adefined volume of reaction mixture, divided by the number of targetnucleic acids within the defined volume of the reaction mixture. In someembodiments, the beads and the target nucleic acids are added to givenamounts of oil and/or aqueous phases to form a reaction mixturecontaining beads, target nucleic acids and oil phases and/or aqueousphases; optionally the ratio is the number of beads added, divided bythe number of target nucleic acids added, to form a defined volume ofthe reaction mixture. In some embodiments, the reaction mixture includesa particular, predefined or preselected ratio of beads-to-target nucleicacids in a defined volume of the reaction mixture. Optionally, thereaction mixture contains a ratio of beads-to-target nucleic acids in avolume of reaction mixture that either includes or lackscompartmentalization. Optionally, the reaction mixture is a singlereaction mixture. In some embodiments, the reaction mixture contains aratio of beads-to-target nucleic acids in a volume that includes fromabout an average of 1 discrete support and an average of 1 targetnucleic acid per 1 mL reaction mixture to an average of about 2 discretesupports: an average of 1 target nucleic acid per 1 mL reaction mixture,or about an average of 2 discrete support and an average of 1 targetnucleic acid per 1 mL reaction mixture to an average of about 5 discretesupports: an average of 1 target nucleic acid per 1 mL reaction mixture.In some embodiments, the reaction mixture includes an averagebeads-to-target nucleic acid ratio, which can be the average number ofbeads divided by the average number of nucleic acids present in adefined volume of the reaction mixture. For example, the reactionmixture optionally includes a ratio of an average of 5 discrete supportsto an average of 1 target nucleic acid per 1 mL reaction mixture (“5:1average beads-to-target nucleic acid ratio”), or a ratio of an averageof 7 discrete supports to an average of 1 target nucleic acid per 1 mL(“7:1 average beads-to-target nucleic acid ratio”), or a ratio of anaverage of 10 discrete supports to an average of 1 target nucleic acidper 1 mL of reaction mixture (“10:1 average beads-to-target nucleic acidratio”). In some embodiments, the reaction mixture includes an averagebead-to-target nucleic acid ratio of about 1, about 5, or about 7, orabout 10, or about 15, or about 25. In some embodiments, the reactionmixture includes an average bead-to-target nucleic acid ratio of fromabout 1 to about 10, or from 1 to 10, from 1 to 15, or from 1 to 25, orfrom about 1 to about 5, or from 1 to 5, or from 5 to 25, or from 5 to15, or from 5 to 10, or from 5 to 7, or from 7 to 15, or from 7 to 10,or from 7 to 25, or from 15 to 25. In some embodiments, the reactionmixture includes a ratio of about 10 discrete supports:1 target nucleicacid per 1 mL of reaction mixture to about 15 discrete supports:1nucleic acid per 1 mL, or a ratio of about 15 discrete supports:1nucleic acid per 1 mL to about 20 discrete supports:1 nucleic acid per 1mL, or a ratio of about 20 discrete supports:1 nucleic acid per 1 mL toabout 25 discrete supports:1 target nucleic acid per 1 mL, or a ratio ofabout 25 discrete supports:1 target nucleic acid per 1 mL to about 30discrete supports:1 nucleic acid per 1 mL, or a ratio of about 30discrete supports:1 target nucleic acid per 1 mL to about 50 discretesupports:1 target nucleic acid per 1 mL of reaction mixture.

Optionally, the reaction mixture contains a ratio of beads-to-targetnucleic acids in a volume of the reaction mixture that includes about0.5 billion discrete supports and about 50 million target nucleic acidsper 1 mL volume of reaction mixture.

Optionally, the reaction mixture contains a ratio of beads-to-targetnucleic acids in a volume of reaction mixture that includes about 1billion discrete supports and about 100 million target nucleic acids per1 mL of reaction mixture.

Optionally, the ratio of the discrete supports to the target nucleicacids can be increased or decreased per 1 mL reaction mixture.

Optionally, the amount of discrete supports can be increased about 2- to10-fold per 1 mL reaction mixture (e.g., increased to about 1-5 billiondiscrete supports per 1 mL reaction mixture).

Optionally, the amount of discrete supports can be increased about 2- to10-fold per 1 mL reaction mixture (e.g., increased to about 2-10 billiondiscrete supports per 1 mL reaction mixture), or increased about 10- to20-fold per 1 mL reaction mixture (e.g., increased to about 10-20billion discrete supports per 1 mL reaction mixture), or increased about20- to 30-fold per 1 mL reaction mixture (e.g., increased to about 20-30billion discrete supports per 1 mL reaction mixture), or increased about30- to 40-fold per 1 mL reaction mixture (e.g., increased to about 30-40billion discrete supports per 1 mL reaction mixture), or increased about40- to 50-fold or more per 1 mL reaction mixture (e.g., increase toabout 40-50 billion discrete supports or more per 1 mL reactionmixture).

Optionally, the amount of discrete supports can be decreased about 2- to10-fold per 1 mL reaction mixture, or decreased about 10- to 25-fold per1 mL reaction mixture, or decreased about 25- to 50-fold per 1 mLreaction mixture.

Optionally, the amount of target nucleic acids can be increased about 2-to about 5-fold per 1 mL reaction mixture (e.g., increased to about100-250 million target nucleic acids per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be increased about 2-to about 5-fold per 1 mL reaction mixture (e.g., increased to about200-500 million target nucleic acids per 1 mL reaction mixture), orincreased about 5- to about 10-fold per 1 mL reaction mixture (e.g.,increased to about 500-1000 million target nucleic acids per 1 mLreaction mixture), or increased about 10- to about 20-fold per 1 mLreaction mixture (e.g., increased to about 1000-2000 million targetnucleic acids per 1 mL reaction mixture), or increased about 20- toabout 50-fold per 1 mL reaction mixture (e.g., increased to about2000-5000 million target nucleic acids per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be increased up to100-fold or more per 1 mL reaction mixture (e.g., increased to about10,000 million target nucleic acids or more per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be decreased about 2-to 10-fold per 1 mL reaction mixture, or decreased about 10- to 25-foldper 1 mL reaction mixture, or decreased about 25- to 50-fold per 1 mLreaction mixture.

In some embodiments, the compositions, as well as related, systems,methods, kits and apparatuses, comprising a single reaction mixturecontaining about 6 billion discrete supports and about 300-800 milliontarget nucleic acids in about 2.4 mL reaction mixture.

In some embodiments, the compositions, as well as related, systems,methods, kits and apparatuses, comprise a single reaction mixturecontaining between about 18-24 billion discrete supports and about 1800million target nucleic acids in a defined volume. Optionally, thedefined volume of the reaction mixture is between 1 mL and 5 mL,typically between 2 and 4 mL, more typically between 2 and 3 mL. In someembodiments, the defined volume is 2.4 mL. Optionally, the reactionmixture includes or lacks compartmentalization.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a plurality of differentpopulations of target nucleic acids, and a plurality of different typesof discrete supports, and (b) forming different amplified populations ofthe target nucleic acids that are attached to the different types ofdiscrete supports. In some embodiments, the methods for synthesizingnucleic acids can be used to amplify nucleic acids.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, foramplifying different target nucleic acids by one or more rounds ofnucleic acid synthesis.

In some embodiments, the plurality of different populations of targetnucleic acids, can bind the plurality of different types of discretesupports. In some embodiments, the different types of discrete supportsinclude different types of capture primers. In some embodiments, theplurality of different populations of target nucleic acids binds thedifferent types of capture primers. In some embodiments, the pluralityof different populations of target nucleic acids, and the plurality ofdifferent types of discrete supports, are provided in a single reactionmixture. In some embodiment, the single reaction mixture comprises anucleic acid amplification reaction mixture. In some embodiments, thesingle reaction mixture includes any one or any combination of: primers(e.g., capture primer, fusion primer, and/or reverse primers), enzymes(e.g., polymerases), accessory proteins (recombinase, recombinaseloading protein, single-stranded binding protein, helicase ortopoisomerase), one or more nucleotides, divalent cations, bindingpartners and/or co-factors. Optionally, the single reaction mixtureprovides compartmentalization, or does not provide compartmentalization.Optionally, the single reaction mixture comprises an emulsion.Optionally, the single reaction mixture can be contained in a singlereaction vessel. In some embodiments, the plurality of differentdiscrete supports includes a plurality of different beads. In someembodiments, the plurality of different types of discrete supportsincludes two, three, four, five, six, seven, eight, nine, ten, eleven,or more different types of discrete supports. In some embodiments, thedifferent amplified populations of the target nucleic acids are formedby conducting any type of nucleic acid amplification reaction, includingPCR, isothermal, rolling circle, or emulsion-based amplification. Insome embodiments, the different amplified populations of the targetnucleic acids are substantially monoclonal. Optionally, the methodfurther comprises sequencing the different amplified populations of thetarget nucleic acids that are attached to the different types ofdiscrete supports.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing at least two differentpopulations of target nucleic acids and at least two differentpopulations of discrete supports and (b) forming at least two differentamplified populations of the target nucleic acids that are attached tothe different types of discrete supports.

In some embodiments, the plurality of different populations of targetnucleic acids, can bind the plurality of different types of discretesupports. In some embodiments, the at least two different populations ofdiscrete supports include different types of capture primers. In someembodiments, the at least two different populations of target nucleicacids selectively binds the different types of capture primers. In someembodiments, the plurality of different populations of target nucleicacids, and the plurality of different types of discrete supports, areprovided in a single reaction mixture. In some embodiment, the singlereaction mixture comprises a nucleic acid amplification reactionmixture. In some embodiments, the single reaction mixture includes anyone or any combination of: primers (e.g., capture primer, fusion primer,and/or reverse primers), enzymes (e.g., polymerases), accessory proteins(recombinase, recombinase loading protein, single-stranded bindingprotein, helicase or topoisomerase), one or more nucleotides, divalentcations, binding partners and/or co-factors. Optionally, the singlereaction mixture provides compartmentalization, or does not providecompartmentalization. Optionally, the single reaction mixture comprisesan emulsion. Optionally, the single reaction mixture can be contained ina single reaction vessel. In some embodiments, the at least twodifferent populations of discrete supports includes at least twodifferent populations of beads. In some embodiments, the at least twodifferent populations of discrete supports includes two, three, four,five, six, seven, eight, nine, ten, eleven, or more different types ofdiscrete supports. In some embodiments, the at least two differentamplified populations of the target nucleic acids are formed byconducting any type of nucleic acid amplification reaction, includingPCR, isothermal, rolling circle, or emulsion-based amplification. Insome embodiments, the at least two different amplified populations ofthe target nucleic acids are substantially monoclonal. Optionally, themethod further comprises sequencing the at least two different amplifiedpopulations of the target nucleic acids that are attached to thedifferent types of discrete supports.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) at least a first population of beads, (ii) at least afirst population of target nucleic acids, (iii) at least a secondpopulation of beads, and (iv) at least a second population of targetnucleic acids; and (b) forming a first and a second amplified populationof the first and second populations of target nucleic acids that areattached to the first and second population of beads, respectively.

In some embodiments, the at least a first population of target nucleicacids can bind the at least a first population of beads. In someembodiments, the at least a second population of target nucleic acidscan bind the at least a second population of beads. In some embodiments,the at least first population of beads includes a first type of captureprimers. In some embodiments, the at least second population of beadsincludes a second type of capture primers. In some embodiments, the atleast first population of target nucleic acids selectively binds thefirst type of capture primers. In some embodiments, the at least secondpopulation of target nucleic acids selectively binds the second type ofcapture primers. In some embodiment, the single reaction mixturecomprises a nucleic acid amplification reaction mixture. In someembodiments, the single reaction mixture includes any one or anycombination of: primers (e.g., capture primer, fusion primer, and/orreverse primers), enzymes (e.g., polymerases), accessory proteins(recombinase, recombinase loading protein, single-stranded bindingprotein, helicase or topoisomerase), one or more nucleotides, divalentcations, binding partners and/or co-factors. Optionally, the singlereaction mixture provides compartmentalization, or does not providecompartmentalization. Optionally, the single reaction mixture comprisesan emulsion. Optionally, the single reaction mixture can be contained ina single reaction vessel. In some embodiments, the single reactionmixture contains two, three, four, five, six, seven, eight, nine, ten,eleven, or more different populations of beads. In some embodiments, thefirst and the second amplified populations of the first and secondpopulations of target nucleic acids are formed by conducting any type ofnucleic acid amplification reaction, including PCR, isothermal, rollingcircle, or emulsion-based amplification. In some embodiments, the firstamplified population of the first population of target nucleic acids issubstantially monoclonal. In some embodiments, the second amplifiedpopulation of the second population of target nucleic acids issubstantially monoclonal. Optionally, the method further comprisessequencing the first and the second amplified populations of targetnucleic acids.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, (ii) a plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids; (b) forminga first amplified population of nucleic acids by amplifying one or moretarget nucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing (i) a single reactionmixture containing a plurality of beads of a first type, the beads ofthe first type including first capture primers, (ii) a plurality ofbeads of a second type, the beads of the second type including secondcapture primers, wherein the first and second capture primers aredifferent, (iii) a first population of target nucleic acids, wherein thefirst population includes at least one target nucleic acid of a firsttype that binds to the first capture primers, and (iv) a secondpopulation of target nucleic acids, wherein the second populationincludes at least one target nucleic acid of a second type that binds tothe second capture primers; (b) forming a first amplified population ofnucleic acids by amplifying one or more target nucleic acids of thefirst population, wherein the first amplified population is attached toone or more beads of the first type; and (c) forming a second amplifiedpopulation of nucleic acids by amplifying one or more target sequencesfrom the second population of target nucleic acids, wherein the secondamplified population is attached to one or more beads of the secondtype.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, formonoclonally amplifying a first target sequence from a first populationof target nucleic acids and a first target sequence from a secondpopulation of target nucleic acids comprising: (a) providing a singlereaction mixture having (i) a plurality of a first type of beadsincluding first capture primers, (ii) a plurality of a second type ofbeads including second capture primers, (iii) a first population oftarget nucleic acids, (iv) a first fusion primer that includes a portionthat is complementary to the first capture primers, (v) a secondpopulation of target nucleic acids, and (vi) a second fusion primer thatincludes a portion that is complementary to the second capture primers;(b) forming a substantially monoclonal population of the first targetsequence from the first population of target nucleic acids on the firsttype of beads using the first fusion primer; and (c) forming asubstantially monoclonal population of the first target sequence fromthe second population of target nucleic acids on the second type ofbeads using the second fusion primer. Optionally, the first and secondcapture primers are different. Optionally, the first population includesat least one target nucleic acid that binds to the first fusion primer.Optionally, the second population includes at least one target nucleicacid that binds to the second fusion primer.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, foramplifying a plurality of different nucleic acid populations in a singlereaction mixture, comprising: (a) providing a single reaction mixtureincluding (i) a first and a second population of nucleic acids, whereinthe first population of nucleic acids contain a first primer bindingsequence, and wherein the second population of nucleic acids contain asecond primer binding sequence, and (ii) a first plurality of beadsincluding a first capture sequence that can bind to the first primerbinding sequence, (iii) a second plurality of beads including a secondcapture sequence that can bind to the second primer binding sequence;and (b) amplifying, within the single reaction mixture, one or morenucleic acids from the first population to form a first amplifiedpopulation, and one or more nucleic acids from the second population toform a second amplified population.

In some embodiments, the sequences of the first and the second primerbinding sequences differ from each other.

In some embodiments, the sequences of the first and second capturesequences are different from each other.

In some embodiments, the first amplified population can be bound to oneor more beads of the first type.

In some embodiments, the second amplified population can be bound to oneor more beads of the second type.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids

In some embodiments, the plurality of beads of the first type includesone or more first type of capture primers.

In some embodiments, the plurality of beads of the second type includesone or more second type of capture primers.

In some embodiments, the sequences of the one or more first captureprimers on the first type of beads are the same or a mixture ofdifferent sequences.

In some embodiments, the sequences of the one or more second captureprimers on the second type of beads are the same or a mixture ofdifferent sequences.

In some embodiments, the sequences of the first capture primers are thesame as the sequences of the second capture primers.

In some embodiments, the sequences of the first capture primers aredifferent from the sequences of the second capture primers.

Optionally, the concentration (e.g., density) of the first and thesecond capture primers is about the same.

In some embodiments, the first population of target nucleic acids canbind the plurality of beads of the first type.

In some embodiments, the first population of target nucleic acidsincludes at least one target nucleic acid of a first type that binds tothe first capture primers.

Optionally, the first population of target nucleic acids includes atleast one target nucleic acid of the first type having a sequence thatis complementary or identical to the sequence of the first captureprimers.

In some embodiments, the first population of target nucleic acidsselectively binds the first type of capture primers.

Optionally, the first population of target nucleic acids includes atleast one target nucleic acid of the first type that binds the firsttype of beads by hybridizing to the first capture primers.

In some embodiments, the second population of target nucleic acids canbind the plurality of beads of the second type.

In some embodiments, the second population of target nucleic acidsincludes at least one target nucleic acid of a second type that binds tothe second capture primers.

Optionally, the second population of target nucleic acids includes atleast one target nucleic acid of the second type having a sequence thatis complementary or identical to the sequence of the second captureprimers.

Optionally, the second population of target nucleic acids binds thesecond capture primers selectively.

Optionally, the second population of target nucleic acids includes atleast one target nucleic acid of the second type that binds the secondtype of beads by hybridizing to the second capture primers.

In some embodiment, the single reaction mixture comprises a nucleic acidamplification reaction mixture.

In some embodiments, the single reaction mixture includes any one or anycombination of: primers (e.g., capture primer, fusion primer, and/orreverse primers), enzymes (e.g., polymerases), accessory proteins(recombinase, recombinase loading protein, single-stranded bindingprotein, helicase or topoisomerase), one or more nucleotides, divalentcations, binding partners and/or co-factors.

Optionally, the single reaction mixture provides compartmentalization,or does not provide compartmentalization.

Optionally, the single reaction mixture comprises an emulsion.Optionally, the emulsion comprises a water-in-oil emulsion.

Optionally, the single reaction mixture can be contained in a singlereaction vessel.

In some embodiments, the single reaction mixture contains two, three,four, five, six, seven, eight, nine, ten, eleven, or more differenttypes of beads, where each different type of beads includes a differenttype of capture primer.

In some embodiments, the first amplified population of nucleic acids isformed by amplifying one or more target nucleic acids of the firstpopulation.

In some embodiments, the forming the first amplified population ofnucleic acids includes hybridizing at least one target nucleic acid fromthe first population of target nucleic acids to the first capture primeron the first type of beads.

Optionally, the forming the first amplified population of nucleic acidsfurther includes extending the first capture primer in atemplate-dependent primer extension reaction.

In some embodiments, the first amplified population of nucleic acids iscovalently attached to one or more beads of the first type.

In some embodiments, the first amplified population of nucleic acids isformed by conducting any type of nucleic acid amplification reaction,including PCR, isothermal, rolling circle, or emulsion-basedamplification.

In some embodiments, the first amplified population of nucleic acids issubstantially monoclonal.

In some embodiments, the second amplified population of nucleic acids isformed by amplifying one or more target nucleic acids of the secondpopulation.

In some embodiments, the forming the second amplified population ofnucleic acids includes hybridizing at least one target nucleic acid fromthe second population of target nucleic acids to the second captureprimer on the second type of beads.

Optionally, the forming the second amplified population of nucleic acidsfurther includes extending the second capture primer in atemplate-dependent primer extension reaction.

In some embodiments, the second amplified population of nucleic acids iscovalently attached to one or more beads of the second type.

In some embodiments, the second amplified population of nucleic acids isformed by conducting any type of nucleic acid amplification reaction,including PCR, isothermal, rolling circle, or emulsion-basedamplification.

In some embodiments, the second amplified population of nucleic acids issubstantially monoclonal.

In some embodiments, the first and the second amplified populations ofnucleic acids are both substantially monoclonal.

Optionally, the method further comprises sequencing the first amplifiedpopulation of nucleic acids. Optionally, the method further comprisessequencing the second amplified population of nucleic acids. Optionally,the first and the second amplified populations of nucleic acids aresequenced in parallel. Optionally, the sequencing includes detecting oneor more nucleotide incorporation byproducts. Optionally, the sequencingincludes detecting hydrogen ions or pyrophosphate.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, (ii) a plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids; (b) forminga first amplified population of nucleic acids by amplifying one or moretarget nucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids, where thesingle reaction mixture does not provide compartmentalization orpartitioning.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, (ii) a plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids; (b) forminga first amplified population of nucleic acids by amplifying one or moretarget nucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids, where thesingle reaction mixture does not provide compartmentalization orpartitioning, and optionally, where the single reaction mixture alsoincludes one or more accessory proteins including recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, the beads of thefirst type being attached to one or more first capture primers, (ii) aplurality of beads of a second type, the beads of the second type beingattached to one or more second capture primers, wherein the first andsecond capture primers are different, (iii) a first population of targetnucleic acids, wherein the first population includes at least twodifferent target nucleic acids that can each bind independently to afirst capture primer, and (iv) a second population of target nucleicacids, wherein the second population includes at least two differenttarget nucleic acids that can each bind independently to a secondcapture primer; (b) forming a first amplified population of nucleicacids by amplifying one or more target nucleic acids of the firstpopulation; and (c) forming a second amplified population of nucleicacids by amplifying one or more target sequences from the secondpopulation of target nucleic acids, where the single reaction mixturedoes not provide compartmentalization or partitioning, and optionally,where the single reaction mixture also includes one or more accessoryproteins including recombinase, recombinase loading protein,single-stranded binding protein, helicase or topoisomerase.

In some embodiments, in the methods for synthesizing nucleic acids, thesingle reaction mixture comprises a single continuous liquid phase. Insome embodiments, the single continuous liquid phase comprises anaqueous phase liquid. In some embodiments, the single continuous liquidphase lacks a hydrophobic phase.

In some embodiments, the single continuous liquid phase does notpartition the first type of beads from the second type of beads. In someembodiments, the single continuous liquid phase does not partition thefirst population of target nucleic acids from the second population oftarget nucleic acids. In some embodiments, the single continuous liquidphase does not partition any of the types of beads from any of the typesof target nucleic acids.

In some embodiments, a single reaction vessel contains a single reactionmixture which includes at least two different types of beads and atleast two different types of target nucleic acids, where the singlereaction mixture does not provide compartmentalization or partitioning.

In some embodiments, the single reaction mixture does not providecompartmentalization, and the first population of target nucleic acidscontains target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the first population of target nucleic acids contains two ormore target nucleic acids that can bind the first plurality of beads.

Optionally, the binding between the first plurality of beads and thefirst population of target nucleic acids comprise selective binding.

Optionally, one or more first type of capture primers are attached tothe first plurality of beads.

Optionally, the first population of target nucleic acids contains two ormore different target nucleic acids that can bind independently to firstcapture primers.

Optionally, first plurality of beads is attached with one or more of afirst type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the first type of capture primers are attached covalently tothe first plurality of beads.

Optionally, the first plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the first plurality of beads do not include any secondcapture primers.

Optionally, the first type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the first population of targetnucleic acids are bound to the first plurality of beads.

Optionally, the target nucleic acids of the first population of targetnucleic acids are bound to the first plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the firstpopulation of target nucleic acids are each hybridized to a firstcapture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has a sequence that is complementaryto at least a region of a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

In some embodiments, the single reaction mixture does not providecompartmentalization, and the second population of target nucleic acidscontains target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the second population of target nucleic acids contains twoor more target nucleic acids that can bind the second plurality ofbeads.

Optionally, the binding between the second plurality of beads and thesecond population of target nucleic acids comprise selective binding.

Optionally, one or more second type of capture primers are attached tothe second plurality of beads.

Optionally, the second population of target nucleic acids contains twoor more different target nucleic acids that can bind independently tosecond capture primers.

Optionally, second plurality of beads is attached with one or more of asecond type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the second type of capture primers are attached covalentlyto the second plurality of beads.

Optionally, the second plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the second plurality of beads do not include any firstcapture primers.

Optionally, the second type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the second population of targetnucleic acids are bound to the second plurality of beads.

Optionally, the target nucleic acids of the second population of targetnucleic acids are bound to the second plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the secondpopulation of target nucleic acids are each hybridized to a secondcapture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has a sequence that is complementaryto at least a region of a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the target nucleic acids in the first population of targetnucleic acids can have the same sequence or a mixture of differentsequences.

Optionally, the target nucleic acids in the second population of targetnucleic acids can have the same sequence or a mixture of differentsequences.

Optionally, at least one target nucleic acid from the first populationof target nucleic acids and at least one target nucleic acid from thesecond population of target nucleic have the same sequence.

Optionally, the first population of target nucleic acids and the secondpopulation of target nucleic acids do not contain any target nucleicacids with the same sequence.

Optionally, the first plurality of beads is attached with a first typeof capture primers, and the second plurality of beads is attached with asecond type of capture primers, and the sequences of the first type ofcapture primers and the second type of capture primers are the same ordiffer.

Optionally, the first plurality of beads includes only capture primersof the first type.

Optionally, the second plurality of beads includes only capture primersof the second type.

Optionally, the first population of beads binds selectively, viahybridization, to the first population of target nucleic acids.

Optionally, the second population of beads binds selectively, viahybridization, to the second population of target nucleic acids.

Optionally, the selective binding between the first population of beadsand the first population of target nucleic acids, and between the secondpopulation of beads and the second population of target nucleic acids,occurs in a single reaction mixture.

Optionally, the selective binding between the first population of beadsand the first population of target nucleic acids, and between the secondpopulation of beads and the second population of target nucleic acids,occurs in a single reaction mixture that provides nocompartmentalization.

In some embodiments, the methods (and related compositions, systems,kits and apparatuses) which comprise a single reaction mixture (e.g.,that does not provide compartmentalization), further includes additionalprimer types, including third, fourth, fifth, or more different primertypes.

In some embodiments, the methods (and related compositions, systems,kits and apparatuses) which comprise a single reaction mixture (e.g.,that does not provide compartmentalization, further includes any one orany combination of: additional primers (e.g., capture primer, fusionprimer, and/or reverse primers), enzymes (e.g., polymerases), accessoryproteins (recombinase, recombinase loading protein, single-strandedbinding protein, helicase or topoisomerase), one or more nucleotides,divalent cations, binding partners and/or co-factors.

Optionally, the enzymes catalyze nucleotide incorporation (e.g.,polymerase).

Optionally, the enzymes comprise accessory proteins.

Optionally, the additional primers include primers in solution orattached to a support (e.g., a bead or particle).

Optionally, the nucleotides comprise natural nucleotides or analogsthereof.

Optionally, the binding partners comprise biotin.

Optionally, the co-factors include salts, cations, ATP, phosphocreatine,magnesium, manganese, and calcium.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction mixture.

Optionally, the single reaction mixture comprises an amplificationreaction mixture.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction vessel.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, (ii) a plurality ofbeads of a second type, (iii) a first population of target nucleicacids, and (iv) a second population of target nucleic acids; (b) forminga first amplified population of nucleic acids by amplifying one or moretarget nucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids, where thesingle reaction mixture provides compartmentalization or partitioning.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturethat includes an emulsion, the single reaction mixture containing (i) aplurality of beads of a first type, (ii) a plurality of beads of asecond type, (iii) a first population of target nucleic acids, and (iv)a second population of target nucleic acids; (b) forming a firstamplified population of nucleic acids by amplifying one or more targetnucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids.

In some embodiments, the single reaction mixture provides at least onecompartment that contains at least two different types of beads and atleast two different types of target nucleic acids. For example, thesingle reaction mixture has at least one compartment that contains (1) afirst plurality of beads of a first type and a second plurality of beadsof a second type, and (2) a first population of target nucleic acids anda second population of target nucleic acids. Optionally, the at leastone compartment can contain additional different types of beads andadditional different types of target nucleic acids.

In some embodiments, in the methods for synthesizing nucleic acids, thesingle reaction mixture contains an emulsion that providescompartmentalization or partitioning.

Optionally, the emulsion comprises a discontinuous hydrophilic phase anda continuous hydrophobic phase.

Optionally, the discontinuous hydrophilic phase is surrounded by thecontinuous hydrophobic phase.

Optionally, the emulsion comprises at least one hydrophilic phasecompartment (e.g., droplet or micro-reactor) surrounded by a continuoushydrophobic phase.

Optionally, the discontinuous hydrophilic phase provides a compartment.

Optionally, the emulsion comprises a plurality of hydrophilic phasedroplets and a continuous hydrophobic phase.

Optionally, the emulsion comprises a plurality of aqueous droplets and acontinuous hydrophobic phase.

Optionally, the emulsion comprises at least one aqueous droplet.

Optionally, the at least one aqueous droplet includes one or more beads.

Optionally, the at least one aqueous droplet includes one or moredifferent target nucleic acids.

Optionally, the emulsion includes at least one aqueous droplet thatincludes one or more beads of the first type, the second type, or beadsof both the first and second type.

Optionally, the emulsion includes at least one aqueous droplet thatincludes one or more different target nucleic acids, including the firstpopulation, the second population, or both the first and secondpopulations of target nucleic acids.

In some embodiments, the emulsion comprises two immiscible liquidphases. In some embodiments, two immiscible liquid phases are mixedtogether to make the emulsion. In some embodiments, one of the liquidphases is dispersed in the other. Optionally, the emulsion comprises amixture of an aqueous liquid and a water-immiscible organic liquid.Optionally, the emulsion comprises at least one anionic, cationic ornon-ionic surfactant. Optionally, the emulsion can have a droplet-typedispersion comprising oil-in-water, water-in-oil, or a bicontinuousmicroemulsion.

In some embodiments, the water immiscible organic liquid comprises anoil. In some embodiments, the oil can be from a natural source,including animal (e.g., tallow or lard), fish (e.g., fish oil), shark,seeds, nuts or plants (e.g., vegetable oils). Optionally, the oil can befrom derived from petroleum, including mineral oils. Optionally, the oilcomprises a fluorochemical oil, polyalphaolefin or ester oil.

In some embodiments, the surfactant includes small molecule surfactants,polymeric surfactants, triblock co-polymer surfactants or non-ionicblock copolymer surfactants. Optionally, the surfactant comprises asorbitan oleate or a silicone surfactant.

Optionally, the hydrophilic phase compartment can contain at least twodifferent types of beads. Optionally, the hydrophilic phase compartmentcan contain at least two different types of target nucleic acids.

Optionally, at least one hydrophilic phase compartment can contain (i) afirst plurality of beads of a first type, (ii) a first population oftarget nucleic acids, (iii) a second plurality of beads of a secondtype, and (iv) a second population of target nucleic acids. Optionally,the at least one hydrophilic phase compartment can contain additionaldifferent types of beads and additional different types of targetnucleic acids. Optionally, the hydrophilic phase compartment can furthercontain any one or any combination of: primers (e.g., capture primer,fusion primer, and/or reverse primers), enzymes (e.g., polymerases),accessory proteins (recombinase, recombinase loading protein,single-stranded binding protein, helicase or topoisomerase), one or morenucleotides, divalent cations, binding partners and/or co-factors.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the first population of target nucleic acids whichinclude target nucleic acids having the same sequence or a mixture ofdifferent sequences.

Optionally, the first population of target nucleic acids contains two ormore target nucleic acids that can bind the first plurality of beads.

Optionally, the binding between the first plurality of beads and thefirst population of target nucleic acids comprise selective binding.

Optionally, the first plurality of beads is attached to one or morefirst type of capture primers.

Optionally, the first population of target nucleic acids contains two ormore different target nucleic acids that can bind independently to firstcapture primers.

Optionally, first plurality of beads is attached with one or more of afirst type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the first type of capture primers are attached covalently tothe first plurality of beads.

Optionally, the first plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the first plurality of beads do not include any secondcapture primers.

Optionally, the first type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the first population of targetnucleic acids are bound to the first plurality of beads.

Optionally, the target nucleic acids of the first population of targetnucleic acids are bound to the first plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the firstpopulation of target nucleic acids are each hybridized to a firstcapture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids is complementary to at least a regionof a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the firstpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the second population of target nucleic acidswhich include target nucleic acids having the same sequence or a mixtureof different sequences.

Optionally, the second population of target nucleic acids contains twoor more target nucleic acids that can bind the second plurality ofbeads.

Optionally, the binding between the second plurality of beads and thesecond population of target nucleic acids comprise selective binding.

In some embodiments, one or more second type of capture primers areattached to the second plurality of beads.

Optionally, the second population of target nucleic acids contains twoor more different target nucleic acids that can bind independently tosecond capture primers.

Optionally, second plurality of beads is attached with one or more of asecond type of capture primers having the same sequence or a mixture ofdifferent sequences.

Optionally, the second type of capture primers are attached covalentlyto the second plurality of beads.

Optionally, the second plurality of beads is attached with one type ofcapture primers having the same sequence.

Optionally, the second plurality of beads do not include any firstcapture primers.

Optionally, the second type of capture primers include at least oneunique identifier sequence.

Optionally, the target nucleic acids in the second population of targetnucleic acids are bound to the second plurality of beads.

Optionally, the target nucleic acids of the second population of targetnucleic acids are bound to the second plurality of beads byhybridization.

Optionally, at least two different target nucleic acids from the secondpopulation of target nucleic acids are each hybridized to a secondcapture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids is complementary to at least a regionof a first or second capture primer.

Optionally, at least one region of a target nucleic acid in the secondpopulation of target nucleic acids has an identical sequence to at leasta region of a first or second capture primer.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains at least one target nucleic acid from the firstpopulation of target nucleic acids and at least one target nucleic acidfrom the second population of target nucleic with the same sequence.

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains the first population of target nucleic acids andthe second population of target nucleic acids which do not include anytarget nucleic acids with the same sequence.

Optionally, the first plurality of beads is attached with a first typeof capture primers, and the second plurality of beads is attached with asecond type of capture primers, and the sequences of the first type ofcapture primers and the second type of capture primers are the same ordifferent.

Optionally, the first plurality of beads includes only capture primersof the first type.

Optionally, the second plurality of beads includes only capture primersof the second type.

Optionally, the first population of beads binds selectively, viahybridization, to the first population of target nucleic acids.

Optionally, the second population of beads binds selectively, viahybridization, to the second population of target nucleic acids.

Optionally, the selective binding occurs in the at least one compartment(e.g., hydrophilic phase compartment).

Optionally, the at least one compartment (e.g., hydrophilic phasecompartment) contains additional primers, including third, fourth,fifth, or more different primer types.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids by (a) providing a single reaction mixturecontaining (i) a plurality of beads of a first type, (ii) a plurality ofbeads of a second type, (iii) a first population of target nucleicacids, (iv) a second population of target nucleic acids, and (v) any oneor any combination of: additional primers (e.g., capture primer, fusionprimer, and/or reverse primers), enzymes (e.g., polymerases), accessoryproteins (recombinase, recombinase loading protein, single-strandedbinding protein, helicase or topoisomerase), one or more nucleotides,divalent cations, binding partners and/or co-factors; (b) forming afirst amplified population of nucleic acids by amplifying one or moretarget nucleic acids of the first population; and (c) forming a secondamplified population of nucleic acids by amplifying one or more targetsequences from the second population of target nucleic acids, where thesingle reaction mixture provides compartmentalization or partitioning.

Optionally, the enzymes catalyze nucleotide incorporation (e.g.,polymerase).

Optionally, the enzymes comprise accessory proteins (e.g., recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase).

Optionally, the additional primers include primers in solution orattached to at least one discrete support.

Optionally, the nucleotides comprise natural nucleotides or analogsthereof.

Optionally, the binding partners comprise biotin.

Optionally, the co-factors include salts, cations, ATP, phosphocreatine,magnesium, manganese, and calcium.

Optionally, the single reaction mixture comprises an amplificationreaction mixture.

Optionally, the first plurality of beads of a first type, the firstpopulation of target nucleic acids, the second plurality of beads of asecond type, and the second population of target nucleic acids, arecontained in a single reaction vessel.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids comprising (a) providing a reaction mixturecontaining a plurality of different template nucleic acids with aplurality of different discrete supports, where the reaction mixtureincludes a ratio of beads-to-target nucleic acids in a volume of thereaction mixture, and (b) amplifying different target nucleic acids withdifferent discrete supports. Optionally, the reaction mixture contains aratio of beads-to-target nucleic acids in a reaction mixture thatincludes or lacks compartmentalization. Optionally, the reaction mixtureis a single reaction mixture.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, where thereaction mixture contains a ratio of beads-to-target nucleic acids in avolume that includes from about an average of 1 discrete support and anaverage of 1 target nucleic acid per 1 mL reaction mixture to an averageof about 2 discrete supports: an average of 1 target nucleic acid per 1mL reaction mixture, or about an average of 2 discrete support and anaverage of 1 target nucleic acid per 1 mL reaction mixture to an averageof about 5 discrete supports: an average of 1 target nucleic acid per 1mL reaction mixture. In some embodiments, the reaction mixture includesan average beads-to-target nucleic acid ratio, which can be the averagenumber of beads divided by the average number of nucleic acids presentin a defined volume of the reaction mixture. For example, the reactionmixture optionally includes a ratio of an average of 5 discrete supportsto an average of 1 target nucleic acid per 1 mL reaction mixture (“5:1average beads-to-target nucleic acid ratio”), or a ratio of an averageof 7 discrete supports to an average of 1 target nucleic acid per 1 mL(“7:1 average beads-to-target nucleic acid ratio”), or a ratio of anaverage of 10 discrete supports to an average of 1 target nucleic acidper 1 mL of reaction mixture (“10:1 average beads-to-target nucleic acidratio”). In some embodiments, the reaction mixture includes an averagebead-to-target nucleic acid ratio of about 1, about 5, or about 7, orabout 10, or about 15, or about 25. In some embodiments, the reactionmixture includes an average bead-to-target nucleic acid ratio of fromabout 1 to about 10, or from 1 to 10, from 1 to 15, or from 1 to 25, orfrom about 1 to about 5, or from 1 to 5, or from 5 to 25, or from 5 to15, or from 5 to 10, or from 5 to 7, or from 7 to 15, or from 7 to 10,or from 7 to 25, or from 15 to 25.

In some embodiments, the reaction mixture includes a ratio of about 10discrete supports:1 target nucleic acid per 1 mL of reaction mixture toabout 15 discrete supports:1 nucleic acid per 1 mL, or a ratio of about15 discrete supports:1 nucleic acid per 1 mL to about 20 discretesupports:1 nucleic acid per 1 mL, or a ratio of about 20 discretesupports:1 nucleic acid per 1 mL to about 25 discrete supports:1 targetnucleic acid per 1 mL, or a ratio of about 25 discrete supports:1 targetnucleic acid per 1 mL to about 30 discrete supports:1 nucleic acid per 1mL, or a ratio of about 30 discrete supports:1 target nucleic acid per 1mL to about 50 discrete supports:1 target nucleic acid per 1 mL ofreaction mixture.

Optionally, the reaction mixture contains a volume of reaction mixtureand a ratio of beads-to-target nucleic acids that includes about 0.5billion discrete supports and about 50 million target nucleic acids per1 mL reaction mixture.

Optionally, the reaction mixture contains a volume of reaction mixtureand a ratio of beads-to-target nucleic acids that includes about 1billion discrete supports and about 100 million target nucleic acids per1 mL reaction mixture.

Optionally, the ratio of the discrete supports to the target nucleicacids can be increased or decreased per 1 mL reaction mixture.

Optionally, the amount of discrete supports can be increased about 2- to10-fold per 1 mL reaction mixture (e.g., increased to about 1-5 billiondiscrete supports per 1 mL reaction mixture).

Optionally, the amount of discrete supports can be increased about 2- to10-fold per 1 mL reaction mixture (e.g., increased to about 2-10 billiondiscrete supports per 1 mL reaction mixture), or increased about 10- to20-fold per 1 mL reaction mixture (e.g., increased to about 10-20billion discrete supports per 1 mL reaction mixture), or increased about20- to 30-fold per 1 mL reaction mixture (e.g., increased to about 20-30billion discrete supports per 1 mL reaction mixture), or increased about30- to 40-fold per 1 mL reaction mixture (e.g., increased to about 30-40billion discrete supports per 1 mL reaction mixture), or increased about40- to 50-fold or more per 1 mL reaction mixture (e.g., increase toabout 40-50 billion discrete supports or more per 1 mL reactionmixture).

Optionally, the amount of discrete supports can be decreased about 2- to10-fold per 1 mL reaction mixture, or decreased about 10- to 25-fold per1 mL reaction mixture, or decreased about 25- to 50-fold per 1 mLreaction mixture.

Optionally, the amount of target nucleic acids can be increased about 2-to about 5-fold per 1 mL reaction mixture (e.g., increased to about100-250 million target nucleic acids per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be increased about 2-to about 5-fold per 1 mL reaction mixture (e.g., increased to about200-500 million target nucleic acids per 1 mL reaction mixture), orincreased about 5- to about 10-fold per 1 mL reaction mixture (e.g.,increased to about 500-1000 million target nucleic acids per 1 mLreaction mixture), or increased about 10- to about 20-fold per 1 mLreaction mixture (e.g., increased to about 1000-2000 million targetnucleic acids per 1 mL reaction mixture), or increased about 20- toabout 50-fold per 1 mL reaction mixture (e.g., increased to about2000-5000 million target nucleic acids per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be increased up to100-fold or more per 1 mL reaction mixture (e.g., increased to about10,000 million target nucleic acids or more per 1 mL reaction mixture).

Optionally, the amount of target nucleic acids can be decreased about 2-to 10-fold per 1 mL reaction mixture, or decreased about 10- to 25-foldper 1 mL reaction mixture, or decreased about 25- to 50-fold per 1 mLreaction mixture.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids comprising (a) providing a single reactionmixture containing about 6 billion discrete supports and about 300-800million target nucleic acids in about 2.4 mL reaction mixture, and (b)amplifying at least some portion of the target nucleic acids bysubjecting the reaction mixture to amplification conditions. Optionally,the ratio of beads-to-target nucleic acids in the reaction mixtureincludes or lacks compartmentalization.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids comprising (a) providing a single reactionmixture containing between 18-24 billion discrete supports and about1800 million target nucleic acids in a defined volume, and (b)amplifying the target nucleic acids in the reaction mixture bysubjecting the reaction mixture to amplification conditions. Optionally,the defined volume of the reaction mixture is between 1 mL and 5 mL,typically between 2 and 4 mL, more typically between 2 and 3 mL. In someembodiments, the defined volume is 2.4 mL. Optionally, the reactionmixture includes or lacks compartmentalization.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least a first plurality of beads of first type and a second pluralityof beads of a second type. One skilled in the art will appreciate thatthe compositions, as well as related, systems, methods, kits andapparatuses, can include a third, fourth, fifth, sixth, seventh, eighth,ninth, tenth, eleventh, or more different plurality of beads.

In some embodiments, the plurality of beads can be solid, or can have anouter surface and an interior surface. The plurality of beads can beporous, semi porous or non-porous. The plurality of beads can havecavitation or pores, or can include three-dimensional scaffolds. In someembodiments, the plurality of beads can be Ion Sphere™ particles (fromIon Torrent, part of Life Technologies, Carlsbad, Calif.).

In some embodiments, the plurality of beads comprises a polymermaterial. For example, the plurality of beads comprise a gel, hydrogelor acrylamide polymers. In some embodiments, the plurality of beads canhave any shape that is spherical, hemispherical, cylindrical,barrel-shaped, toroidal, rod-like, disc-like, conical, triangular,cubical, polygonal, tubular, wire-like or irregular.

In some embodiments, the beads can be any size that can fit into areaction chamber. For example, the beads can be small enough to fit onebead in a reaction chamber. In some embodiments, the beads can be smallenough so that more than one bead can fit in a reaction chamber. In someembodiments, the smallest cross-sectional length of a bead (e.g.,diameter) can be about 50 microns or less, or about 10 microns or less,or about 3 microns or less, approximately 1 micron or less,approximately 0.5 microns or less, e.g., approximately 0.1, 0.2, 0.3, or0.4 microns, or smaller (e.g., under 1 nanometer, about 1-10 nanometer,about 10-100 nanometers, or about 100-500 nanometers).

In some embodiments, the beads can be attached with one or moredifferent capture primers (e.g., oligonucleotides). In some embodiments,the beads can be attached with a plurality of one capture primers havingthe same sequence, or can be attached a plurality of two or moredifferent capture primers having different sequences. In someembodiments, the beads can be attached with a plurality of at least1,000 oligonucleotide primers, or about 1,000-10,000 oligonucleotideprimers, or about, 10,000-50,000 oligonucleotide primers, or about50,000-75,000 oligonucleotide primers, or about 75,000-100,000oligonucleotide primers, or more.

In some embodiments, the exterior bead surface can be attached with oneor more capture primers. In some embodiments, the exterior bead surfaceand interior scaffolds of the beads, can be attached with one or morecapture primers. A bead surface (including the interior scaffold) can becoated with an acrylamide, carboxylic or amine compound for attaching anucleic acid (e.g., capture primer). In some embodiments, anamino-modified capture primer can be attached to a bead surface that iscoated with a carboxylic acid. In some embodiments, an amino-modifiedcapture primer can be reacted with ethyl(dimethylaminopropyl)carbodiimide (EDC) or EDAC for attachment to a carboxylic acid coatedsurface (with or without N-hydoxysuccinimide (NHS)). A capture primercan be immobilized to an acrylamide compound coating on a bead surface.Beads can be coated with an avidin-like compound (e.g., streptavidin)for binding biotinylated capture primers.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingone or more capture primers attached to a bead. In some embodiments, thecompositions, as well as related, systems, methods, kits andapparatuses, comprise a first and second capture primer. In someembodiments, the compositions, as well as related, systems, methods,kits and apparatuses, comprise additional capture primers, including athird, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, ormore different capture primers. In some embodiments, the differentcapture primers have different nucleotide sequences. In someembodiments, the capture primers comprise polymers ofdeoxyribonucleotides, ribonucleotides, and/or analogs thereof. In someembodiments, the capture primers comprise naturally-occurring,synthetic, recombinant, cloned, amplified, or unamplified forms. In someembodiments, the capture primers comprise DNA, cDNA, RNA, chimericRNA/DNA, or nucleic acid analogs. In some embodiments, the captureprimers comprise a random or degenerate sequence. In some embodiments,at least one portion of the capture primers comprises a sequence thatcan hybridize with at least one portion of a target nucleic acid, or anadaptor joined to a target nucleic acid. In some embodiments, at leastone portion of the capture primers comprises a sequence that canhybridize with at least one portion of a fusion primer. In someembodiments, at least one portion of the capture primers comprises asequence that is identical or is complementary to a portion of a targetnucleic acid, an adaptor, or a fusion primer. In some embodiments, thecapture primers comprise single-stranded oligonucleotides. In someembodiments, the 5′ or 3′ end of the one or more capture primers can beattached to the bead.

In some embodiments, the 3′ end of the capture primer is extendible in aprimer extension reaction. Optionally, the 3′ end of the capture primerincludes a 3′OH group. In some embodiments, the capture primer has ablocking moiety that prevents extension in a primer extension reaction.

In some embodiments, the capture primers can be any length, includingabout 2-100 nucleotides, or about 5-10 nucleotides, or about 10-25nucleotides, or about 25-40 nucleotides, or about 40-55 nucleotides, orabout 55-70 nucleotides, or about 70-85 nucleotides, or about 85-100nucleotides, or longer.

In some embodiments, the capture primers include at least one linkage orbase that is resistant to degradation by an exonuclease or endonuclease.For example, the fusion primers and reverse amplification primers caninclude at least one phosphorothioate linkage or a 3′-3′ end linkage forexonuclease resistance, or at least one 2′ fluoro or 2′O-methylmodification for endonuclease resistance.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, that also include atleast one third primer. At least one portion of the third primerincludes a sequence that can hybridize to at least one region of thetarget nucleic acids in the first population of target nucleic acids, orcan hybridize to at least one region of a complementary sequence of thetarget nucleic acids in the first population of target nucleic acids. Insome embodiments, the third primer includes a sequence that canhybridize to at least one region of the target nucleic acids in thesecond population of target nucleic acids, or can hybridize to at leastone region of a complementary sequence of the target nucleic acids inthe second population of target nucleic acids.

Optionally, the third primer is attached to a bead of the first type orthe second type, or is attached to any bead. Optionally, the attachmentis covalent.

Optionally, the third primer is a solution phase primer (e.g., is notattached to any bead).

Optionally, the third primer comprises a reverse primer (see 150 in FIG.1).

Optionally, the third primer comprises a reverse amplification primer.

Optionally, the third primer includes a universal priming sequence orsite.

Optionally, the third primer includes at least one unique identifiersequence.

Optionally, the third primer includes a binding partner.

Optionally, the binding partner comprises biotin.

Optionally, the 5′ end of the third primer can include a sequence thatis not contained in, or is not complementary to, a sequence in thetarget nucleic acids in the first or the second population of targetnucleic acids. For example, the third primer can be a tailed primer.

In some embodiments, the third primer comprises polymers ofdeoxyribonucleotides, ribonucleotides, and/or analogs thereof. In someembodiments, the third primer comprises naturally-occurring, synthetic,recombinant, cloned, amplified, or unamplified forms. In someembodiments, the third primer comprises DNA, cDNA, RNA, chimericRNA/DNA, or nucleic acid analogs. In some embodiments, the third primercomprises a random or degenerate sequence. In some embodiments, at leastone portion of a third primer comprises a sequence that can hybridizewith at least one portion of a target nucleic acid, or an adaptor joinedto a target nucleic acid, a capture primer, or a fusion primer. In someembodiments, at least one portion of the third primer comprises asequence that is identical or is complementary to a portion of a targetnucleic acid, an adaptor, a capture primer, or a fusion primer. In someembodiments, the third primer comprises single-strandedoligonucleotides.

In some embodiments, the 3′ end of the third primers is extendible in aprimer extension reaction. Optionally, the 3′ end of the third primerincludes a 3′OH group. In some embodiments, the third primer has ablocking moiety that prevents extension in a primer extension reaction.

In some embodiments, the third primer can be any length, including about2-100 nucleotides, or about 5-10 nucleotides, or about 10-25nucleotides, or about 25-40 nucleotides, or about 40-55 nucleotides, orabout 55-70 nucleotides, or about 70-85 nucleotides, or about 85-100nucleotides, or longer.

In some embodiments, the third primers include at least one linkage orbase that is resistant to degradation by an exonuclease or endonuclease.For example, the fusion primers and reverse amplification primers caninclude at least one phosphorothioate linkage or a 3′-3′ end linkage forexonuclease resistance, or at least one 2′ fluoro or 2′O-methylmodification for endonuclease resistance.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, that also include aplurality of different fusion primers that comprise complementary oridentical sequences of a capture primer (or a portion thereof) and atarget nucleic acid (or a portion thereof). For example, thecompositions (and related methods, systems, kits and apparatuses)includes a plurality of a first fusion primer (see 160 of FIG. 2)comprising sequences that are complementary or identical to at least aportion of a first capture primer (see 110 of FIG. 2), and at least aportion of a first target nucleic acid (see 120 and 140 of FIG. 2). Thecompositions (and related methods, systems, kits and apparatuses)includes a plurality of a second fusion primer (see 260 of FIG. 2)comprising sequences that are complementary or identical to at least aportion of a second capture primer (see 210 of FIG. 2), and at least aportion of a second target nucleic acid (see 220 and 240 of FIG. 2).

The compositions (and related methods, systems, kits and apparatuses)can further include a plurality of a third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, or more different fusionprimers.

In some embodiments, the fusion primers comprise polymers ofdeoxyribonucleotides, ribonucleotides, and/or analogs thereof. In someembodiments, the fusion primers comprise naturally-occurring, synthetic,recombinant, cloned, amplified, or unamplified forms. In someembodiments, the fusion primers comprise DNA, cDNA, RNA, chimericRNA/DNA, or nucleic acid analogs. In some embodiments, the fusionprimers comprise a random or degenerate sequence. In some embodiments,at least one portion of the fusion primers have nucleotide sequencesthat are different compared to other fusion primers. In someembodiments, at least one portion of a fusion primer comprises asequence that can hybridize with at least one portion of a targetnucleic acid, or an adaptor joined to a target nucleic acid, or acapture primer. In some embodiments, at least one portion of the captureprimers comprises a sequence that is identical or is complementary to aportion of a target nucleic acid, an adaptor, or a capture primer. Insome embodiments, the fusion primers comprise single-strandedoligonucleotides.

In some embodiments, the fusion primers can be any length, includingabout 2-100 nucleotides, or about 5-10 nucleotides, or about 10-25nucleotides, or about 25-40 nucleotides, or about 40-55 nucleotides, orabout 55-70 nucleotides, or about 70-85 nucleotides, or about 85-100nucleotides, or longer.

In some embodiments, the plurality of first fusion primers, comprise afirst sequence that is complementary or identical to at least aregion/portion of the first capture primers.

Optionally, the plurality of first fusion primers further include asecond sequence that is complementary or identical to at least aregion/portion of a target nucleic acid in the first population oftarget nucleic acids (see 160 of FIG. 2).

Optionally, the second sequence that is included in the plurality offirst fusion primers is complementary to at least one adaptor on atarget nucleic acid (see 160 of FIG. 2).

Optionally, the first fusion primer includes at least one uniqueidentifier sequence.

Optionally, the first fusion primer is attached to a bead. Optionally,the attachment is covalent.

Optionally, the first fusion primer is a solution phase primer (e.g., isnot attached to any bead).

Optionally, the first fusion primer can include a sequence that iscomplementary to a capture primer and a sequence that is complementaryto a portion of a target nucleic acid (e.g., an adaptor sequence) (see160 of FIG. 2). The first fusion primer can be employed to amplify atarget nucleic acid that lacks a sequence that will bind a captureprimer on a bead. Optionally, the first fusion primer is a solubleprimer and is not attached to a support.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses) also includes a second fusion primer, comprising afirst sequence that is complementary or identical to at least aregion/portion of the second capture primers.

Optionally, the second fusion primer further includes a second sequencethat is complementary or identical to at least a region/portion of atarget nucleic acid in the second population of target nucleic acids(see 260 of FIG. 2).

Optionally, the second sequence that is included in the plurality ofsecond fusion primers is complementary to at least one adaptor on atarget nucleic acid (see 160 of FIG. 2).

Optionally, the second fusion primer includes at least one uniqueidentifier sequence.

Optionally, the second fusion primer is attached to a bead. Optionally,the attachment is covalent.

Optionally, the second fusion primer is a solution phase primer (e.g.,is not attached to any bead).

Optionally, the second fusion primer can include a sequence that iscomplementary to a capture primer and a sequence that is complementaryto a portion of a target nucleic acid (e.g., an adaptor sequence) (see260 of FIG. 2). The second fusion primer can be employed to amplify atarget nucleic acid that lacks a sequence that will bind a captureprimer on a bead. Optionally, the second fusion primer is a solubleprimer and is not attached to a support.

In some embodiments, the fusion primers include at least one linkage orbase that is resistant to degradation by an exonuclease or endonuclease.For example, the fusion primers and reverse amplification primers caninclude at least one phosphorothioate linkage or a 3′-3′ end linkage forexonuclease resistance, or at least one 2′ fluoro or 2′O-methylmodification for endonuclease resistance.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least a first population of target nucleic acids and a secondpopulation of target nucleic acids. One skilled in the art willappreciate that the compositions, as well as related, systems, methods,kits and apparatuses, can include a third, fourth, fifth, sixth,seventh, eighth, ninth, tenth, eleventh, or more different populationsof nucleic acids. In some embodiments, the target nucleic acids comprisesingle-stranded or double-stranded polynucleotides, or a mixture ofboth. In some embodiments, the target nucleic acids in a population oftarget nucleic acids, include polynucleotides having the same ordifferent sequences. In some embodiments, the target nucleic acids in apopulation of target nucleic acids includes polynucleotides having thesame or different lengths. In some embodiments, a population of targetnucleic acids can have about 2-10, or about 10-50, or about 50-100, orabout 100-500, or about 500-1,000, or about 1,000-5,000, or about10³-10⁶, or about 10⁶-10¹⁰ or more different target nucleic acidmolecules. In some embodiments, the target nucleic acids comprisepolymers of deoxyribonucleotides, ribonucleotides, and/or analogsthereof. In some embodiments, the target nucleic acids comprisenaturally-occurring, synthetic, recombinant, cloned, amplified,unamplified or archived (e.g., preserved) forms. In some embodiments,the target nucleic acids comprise DNA, cDNA, RNA, RNA/DNA, and nucleicacid analogs.

In some embodiments, two or more target nucleic acids in a population oftarget nucleic acids comprise nucleic acids having one or both endsjoined to a nucleic acid adaptor. For example, the first end of a targetnucleic acid can be joined to a first nucleic acid adaptor. Optionally,the second end of the target nucleic acid can be joined to a secondnucleic acid adaptor. The first and second adaptors can have the same ordifferent sequence. In some embodiments, at least a portion of the firstor second nucleic acid adaptor can hybridize to the capture primer,fusion primer, reverse primer, amplification primer or sequencingprimers.

In some embodiments, target nucleic acids in the population of targetnucleic acids can be compatible for use in any type of sequencingplatform including chemical degradation, chain-termination,sequence-by-synthesis, pyrophosphate, massively parallel, ion-sensitive,and single molecule sequencing platforms.

In some embodiments, at least one target nucleic acid in the first andthe second population of target nucleic acids include one or moreadaptor sequences.

In some embodiments, the target nucleic acids in the first population oftarget nucleic acids include a first adaptor sequence.

Optionally, at least a portion of the first adaptor sequence (see 120 ofFIG. 1) is complementary or identical to a sequence within the firsttype of capture primer (see 110 of FIG. 1).

Optionally, the target nucleic acids in the first population of targetnucleic acids further include a second adaptor sequence.

Optionally, at least a portion of the second adaptor sequence (see 130of FIG. 1) is complementary or identical to the third primer (e.g.,reverse primer) (see 150 of FIG. 1).

Optionally, the target nucleic acids in the first population of targetnucleic acids include a first adaptor and a second adaptor having thesame or different sequences.

In some embodiments, the target nucleic acids in the second populationof target nucleic acids include a third adaptor sequence.

Optionally, at least a portion of the third adaptor sequence (see 220 ofFIG. 1) is complementary or identical to a sequence within the secondtype of capture primer (see 210 of FIG. 1).

Optionally, the target nucleic acids in the second population of targetnucleic acids further include a fourth adaptor sequence.

Optionally, at least a portion of the fourth adaptor sequence (see 230of FIG. 1) is complementary or identical to the third primer (e.g.,reverse primer) (see 250 of FIG. 1).

Optionally, the target nucleic acids in the second population of targetnucleic acids include a third adaptor and a fourth adaptor having thesame or different sequences.

Optionally, the first adaptors of the first population of target nucleicacids and the third adaptors of the second population of target nucleicacids have the same or different sequences.

Optionally, the second adaptors of the first population of targetnucleic acids and the fourth adaptors of the second population of targetnucleic acids have the same or different sequences.

Optionally, the second and fourth adaptors comprise a sequence that is auniversal sequence among the first and second populations of targetnucleic acids.

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include at least one unique identifier sequence.

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include a sequence that is identical orcomplementary to an amplification primer binding sequence.

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include a sequence that is identical orcomplementary to a sequencing primer binding sequence

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include a binding partner.

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include a sequence that is identical orcomplementary to an amplification primer binding sequence.

Optionally, any one or any combination of the first, second, thirdand/or fourth adaptors include a sequence that is identical orcomplementary to a sequencing primer binding sequence.

Optionally, any one or any combination of the first capture primer,second capture primer, first fusion primer, second fusion primer and/orthird primer (e.g., reverse primer) includes a sequence that isidentical or complementary to an amplification primer binding sequence.

Optionally, any one or any combination of the first capture primer,second capture primer, first fusion primer, second fusion primer and/orthird primer (e.g., reverse primer) includes a sequence that isidentical or complementary to a sequencing primer binding sequence.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingtarget nucleic acids and at least one adaptor.

In some embodiments, one or more adaptors can be joined to the targetnucleic acid by ligation. In some embodiments, a tailed amplificationprimer can be used in a primer extension reaction (e.g., PCR reaction)to append one or more adaptors to a target nucleic acid, where thetailed amplification primer includes the sequence of one or moreadaptors. For example, a tailed primer includes a first or secondconversion primer (see 170 a/b and 270 a/b in FIG. 3A) which includes atleast one portion having a sequence that is complementary or identicalto a portion of an adaptor (e.g., see 120 and 220 in FIG. 3A) that isjoined to a target nucleic acid (e.g., see 140 and 240 in FIG. 3A).Optionally, a first or second conversion primer can be used in a primerextension reaction to append a portion of the conversion primer to thetarget nucleic acid, or to the first or third adaptor. In anotherexample, a tailed primer includes a first or second fusion primer (see160 and 260 in FIGS. 2 and 3B) which includes at least one portionhaving a sequence that is complementary or identical to a portion of anadaptor (e.g., see 120 and 220 in FIG. 2, or 170 a and 270 a in FIG. 3B)that is joined to a target nucleic acid (e.g., see 140 and 240 in FIGS.2 and 3B). In some embodiments, a tailed primer includes a first orsecond fusion primer (see 160 and 260 in FIGS. 2 and 3B) which includesat least one portion having a sequence that is not complementary or isnot identical to a portion of an adaptor (e.g., see 120 and 220 in FIG.2, or 170 a and 270 a in FIG. 3B) that is joined to a target nucleicacid (e.g., see 140 and 240 in FIGS. 2 and 3B). Optionally, a first orsecond fusion primer can be used in a primer extension reaction toappend a portion of the fusion primer to the target nucleic acid, or tothe first or third adaptor.

In some embodiments, the adaptor comprises a nucleic acid, includingDNA, RNA, RNA/DNA molecules, or analogs thereof. In some embodiments,the adaptor can include one or more deoxyribonucleoside orribonucleoside residues. In some embodiments, the adaptor can besingle-stranded or double-stranded nucleic acids, or can includesingle-stranded and/or double-stranded portions. In some embodiments,the adaptor can have any structure, including linear, hairpin, forked(Y-shaped), or stem-loop.

In some embodiments, the adaptor can have any length, including fewerthan 10 bases in length, or about 10-20 bases in length, or about 20-50bases in length, or about 50-100 bases in length, or longer.

In some embodiments, the adaptor can have any combination of bluntend(s) and/or sticky end(s). In some embodiments, at least one end ofthe adaptor can be compatible with at least one end of a nucleic acidfragment. In some embodiments, a compatible end of the adaptor can bejoined to a compatible end of a nucleic acid fragment. In someembodiments, the adaptor can have a 5′ or 3′ overhang end.

In some embodiments, the adaptor can have a 5′ or 3′ overhang tail. Insome embodiments, the tail can be any length, including 1-50 or morenucleotides in length.

In some embodiments, the adaptor can include an internal nick. In someembodiments, the adaptor can have at least one strand that lacks aterminal 5′ phosphate residue. In some embodiments, the adaptor lackinga terminal 5′ phosphate residue can be joined to a nucleic acid fragmentto introduce a nick at the junction between the adaptor and the nucleicacid fragment.

In some embodiments, the adaptor can include a nucleotide sequence thatis identical or complementary to any portion of a capture primer, fusionprimer, reverse primer, amplification primer, or a sequencing primer.

In some embodiments, the adaptor can include identification sequences,such as for example, a uniquely identifiable sequence (e.g., barcodesequence). In some embodiments, a barcoded adaptor can be used forconstructing a multiplex library of target nucleic acids. In someembodiments, the barcoded adaptors can be appended to a target nucleicacid and used for sorting or tracking the source of the target nucleicacid. In some embodiments, one or more barcode sequences can allowidentification of a particular adaptor among a mixture of differentadaptors having different barcodes sequences. For example, a mixture caninclude 2, 3, 4, 5, 6, 7-10, 10-50, 50-100, 100-200, 200-500, 500-1000,or more different adaptors having unique barcode sequences.

In some embodiments, the adaptor can include degenerate sequences. Insome embodiments, the adaptor can include one or more inosine residues.

In some embodiments, the adaptor can include at least one scissilelinkage. In some embodiments, the scissile linkage can be susceptible tocleavage or degradation by an enzyme or chemical compound. In someembodiments, the adaptor can include at least one phosphorothiolate,phosphorothioate, and/or phosphoramidate linkage.

In some embodiments, the adaptor can include any type of restrictionenzyme recognition sequence, including type I, type II, type IIs, typeIIB, type III, type IV restriction enzyme recognition sequences, orrecognition sequences having palindromic or non-palindromic recognitionsequences.

In some embodiments, the adaptor can include a cell regulationsequences, including a promoter (inducible or constitutive), enhancers,transcription or translation initiation sequence, transcription ortranslation termination sequence, secretion signals, Kozak sequence,cellular protein binding sequence, and the like.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingone or more polymerases. In some embodiments, the compositions (andrelated methods, systems, kits and apparatuses) includes one type, or amixture of different types of polymerases. In some embodiments, thepolymerase includes any enzyme, or fragment or subunit of thereof, thatcan catalyze polymerization of nucleotides and/or nucleotide analogs. Insome embodiments, the polymerase requires a nucleic acid having anextendible 3′ end. For example, the polymerase can require a terminal 3′OH of a nucleic acid primer to initiate nucleotide polymerization.

The polymerase comprises any enzyme that can catalyze the polymerizationof nucleotides (including analogs thereof) into a nucleic acid strand.Typically but not necessarily such nucleotide polymerization can occurin a template-dependent fashion. In some embodiments, the polymerase canbe a high fidelity polymerase. Such polymerases can include withoutlimitation naturally occurring polymerases and any subunits andtruncations thereof, mutant polymerases, variant polymerases,recombinant, fusion or otherwise engineered polymerases, chemicallymodified polymerases, synthetic molecules or assemblies, and anyanalogs, derivatives or fragments thereof that retain the ability tocatalyze such polymerization. Optionally, the polymerase can be a mutantpolymerase comprising one or more mutations involving the replacement ofone or more amino acids with other amino acids, the insertion ordeletion of one or more amino acids from the polymerase, or the linkageof parts of two or more polymerases. The term “polymerase” and itsvariants, as used herein, also refers to fusion proteins comprising atleast two portions linked to each other, where the first portioncomprises a peptide that can catalyze the polymerization of nucleotidesinto a nucleic acid strand and is linked to a second portion thatcomprises a second polypeptide, such as, for example, a reporter enzymeor a processivity-enhancing domain. Typically, the polymerase comprisesone or more active sites at which nucleotide binding and/or catalysis ofnucleotide polymerization can occur. In some embodiments, the polymeraseincludes or lacks other enzymatic activities, such as for example, 3′ to5′ exonuclease activity or 5′ to 3′ exonuclease activity. In someembodiments, the polymerase can be isolated from a cell, or generatedusing recombinant DNA technology or chemical synthesis methods. In someembodiments, the polymerase can be expressed in prokaryote, eukaryote,viral, or phage organisms. In some embodiments, the polymerase can bepost-translationally modified proteins or fragments thereof.

In some embodiments, the polymerase can be a DNA polymerase and includewithout limitation bacterial DNA polymerases, eukaryotic DNApolymerases, archaeal DNA polymerases, viral DNA polymerases and phageDNA polymerases.

In some embodiments, the polymerase can be a replicase, DNA-dependentpolymerase, primases, RNA-dependent polymerase (including RNA-dependentDNA polymerases such as, for example, reverse transcriptases), athermo-labile polymerase, or a thermo-stable polymerase. In someembodiments, the polymerase can be any Family A or B type polymerase.Many types of Family A (e.g., E. coli Pol I), B (e.g., E. coli Pol II),C (e.g., E. coli Pol III), D (e.g., Euryarchaeotic Pol II), X (e.g.,human Pol beta), and Y (e.g., E. coli UmuC/DinB and eukaryoticRAD30/xeroderma pigmentosum variants) polymerases are described inRothwell and Watsman 2005 Advances in Protein Chemistry 71:401-440. Insome embodiments, a polymerase can be a T3, T5, T7, or SP6 RNApolymerase.

In some embodiments, the polymerase comprises a heat-stable orheat-labile polymerase. In some embodiments, the polymerase comprises alow fidelity or high fidelity polymerase.

In some embodiment, the polymerase can lack 5′-3′ exonuclease activity.In some embodiments, the polymerase can have strand-displacementactivity.

In some embodiments, the archaeal DNA polymerase, can be, withoutlimitation, a thermostable or thermophilic DNA polymerase such as, forexample: a Bacillus subtilis (Bsu) DNA polymerase I large fragment; aTherms aquaticus (Taq) DNA polymerase; a Therms filiformis (Tfi) DNApolymerase; a Phi29 DNA polymerase; a Bacillus stearothermophilus (Bst)DNA polymerase; a Thermococcus sp. 9° N-7 DNA polymerase; a Bacillussmithii (Bsm) DNA polymerase large fragment; a Thermococcus litoralis(Tli) DNA polymerase or Vent™ (exo-) DNA polymerase (from New EnglandBiolabs); or “Deep Vent” (exo-) DNA polymerase (New England Biolabs). Insome embodiments, the polymerase comprises E. coli large fragment DNApolymerase I (e.g., Klenow).

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least one accessory protein. In some embodiments, the accessoryprotein can bind single-stranded or double-stranded nucleic acids.Optionally, the accessory protein can mediate loading other proteins(e.g., recombinase) onto a nucleic acid. Optionally, the accessoryprotein can unwind nucleic acid substrates, relax nucleic acids, resolvenucleic acid structures, hydrolyze nucleic acids (e.g., nuclease),disassemble complexes of nucleic acids and proteins, or disassemblenucleic acid structures. Optionally, the accessory protein can partiallyor fully denature a double-stranded first or second target nucleic acid.Optionally, the accessory protein can catalyze strand invasion orunwinding. Optionally, the accessory protein comprises a sliding clampprotein. Optionally, the accessory protein can mediate or catalyze itsrespective activity in a sequence-specific or sequence-independentmanner.

In some embodiments, an accessory protein comprises a multimeric proteincomplex. Optionally, the multimeric protein complex comprises 2, 3, 4,5, 6, 7, 8, or more subunits. Optionally, the multimeric accessoryprotein complex comprises a homo-meric or hetero-meric protein complex.

In some embodiments, the accessory protein comprises wild-type, mutant,recombinant, fusion, or fragments thereof.

In some embodiments, the accessory proteins can originate from anybacteriophage including a myoviral phage. The accessory proteins canoriginate from bacteriophage T2, T4, T5 or T7. The accessory proteinscan originate from any prokaryote, bacteria (e.g., E. coli), eukaryote,or mammal (e.g., human).

In some embodiments, the accessory proteins comprise a single-strandedbinding protein including myoviral gp32 (e.g., T4 or RB69), Sso SSB fromSulfolobus solfataricus, MjA SSB from Methanococcus jannaschii, or E.coli SSB protein.

In some embodiments, the single reaction mixture comprises a mixture ofdifferent accessory proteins that originate from the same or differentspecies. Optionally, the single reaction mixture comprises a mixture ofdifferent accessory proteins that originate from the same or differentspecies as a recombinase enzyme.

In some embodiment, the accessory protein comprises a single-strandedbinding protein (e.g., SSB or gp32 protein), recombinase (e.g., recA oruvsX), recombinase loading protein (e.g., uvsY protein), helicase (e.g.,uvsW protein), or topoisomerase.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least one accessory protein. In some embodiments, the accessoryprotein comprises an enzyme that catalyzes homologous recombination.Optionally, the enzyme that catalyzes homologous recombination can forma nucleoprotein complex by binding a single-stranded oligonucleotide(e.g., a primer). Optionally, as part of a nucleoprotein complex, thehomologous recombination enzyme can bind a homologous portion of atleast one strand of a double-stranded target nucleic acid. Optionally,the homologous recombination enzyme can catalyze strand unwinding.Optionally, the homologous portion of the target nucleic acid canhybridize to at least a portion of the single-stranded oligonucleotide.Optionally, the homologous portion of the target nucleic acid can bepartially or completely complementary to at least a portion of thesingle-stranded oligonucleotide.

In some embodiments, the accessory protein can catalyze strand invasionby forming a nucleoprotein complex and binding to a homologous portionof a double-stranded target nucleic acid to form a recombinationintermediate having a triple-strand structure (e.g., D-loop formation).

In some embodiments, the accessory protein comprises a recombinaseenzyme.

In some embodiments, the recombinase can form a nucleoprotein complex bybinding a first primer (e.g., first capture primer). Optionally, thenucleoprotein complex further includes a first target nucleic acid,where a portion of the first primer hybridizes to a portion of the firsttarget nucleic acid. Optionally, the first target nucleic acid comprisesa double-stranded polynucleotide molecule. Optionally, the recombinasecan partially or fully denature the double-stranded first target nucleicacid.

In some embodiments, the recombinase can form a nucleoprotein complex bybinding a second primer (e.g., second capture primer). Optionally, thenucleoprotein complex further includes a second target nucleic acid,where a portion of the second primer hybridizes to a portion of thesecond target nucleic acid. Optionally, the second target nucleic acidcomprises a double-stranded polynucleotide molecule. Optionally, therecombinase can partially or fully denature the double-stranded secondtarget nucleic acid.

In some embodiments, the recombination enzyme comprises at least aportion of a recombinase enzyme from any organism, includingbacteriophage T4 (e.g., usvX), Escherichia coli (e.g., recA), or human(e.g., RAD51) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos.5,273,881 and 5,670,316 both to Sena, and U.S. Pat. Nos. 7,270,981,7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000,8,062,850, and 8,071,308).

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingone or more nucleotides. In some embodiments, the compositions (andrelated methods, systems, kits and apparatuses) includes one type, or amixture of different types of nucleotides. A nucleotide comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase. Suchnucleotides include not only naturally occurring nucleotides but alsoany analogs, regardless of their structure, that can bind selectivelyto, or can be polymerized by, a polymerase. While naturally occurringnucleotides typically comprise base, sugar and phosphate moieties, thenucleotides of the present disclosure can include compounds lacking anyone, some or all of such moieties. In some embodiments, the nucleotidecan optionally include a chain of phosphorus atoms comprising three,four, five, six, seven, eight, nine, ten or more phosphorus atoms. Insome embodiments, the phosphorus chain can be attached to any carbon ofa sugar ring, such as the 5′ carbon. The phosphorus chain can be linkedto the sugar with an intervening O or S. In some embodiments, one ormore phosphorus atoms in the chain can be part of a phosphate grouphaving P and O. In some embodiments, the phosphorus atoms in the chaincan be linked together with intervening O, NH, S, methylene, substitutedmethylene, ethylene, substituted ethylene, CNH₂, C(O), C(CH₂), CH₂CH₂,or C(OH)CH₂R (where R can be a 4-pyridine or 1-imidazole). In someembodiments, the phosphorus atoms in the chain can have side groupshaving O, BH₃, or S. In the phosphorus chain, a phosphorus atom with aside group other than O can be a substituted phosphate group. In thephosphorus chain, phosphorus atoms with an intervening atom other than Ocan be a substituted phosphate group. Some examples of nucleotideanalogs are described in Xu, U.S. Pat. No. 7,405,281.

Some examples of nucleotides that can be used in the disclosedcompositions (and related methods, systems, kits and apparatuses)include, but are not limited to, ribonucleotides, deoxyribonucleotides,modified ribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, modified peptide nucleotides,metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof. In some embodiments, a nucleotide can include apurine or pyrimidine base, including adenine, guanine, cytosine, thymineor uracil. In some embodiments, a nucleotide includes dATP, dGTP, dCTP,dTTP and dUTP.

In some embodiments, the nucleotide is unlabeled. In some embodiments,the nucleotide comprises a label and referred to herein as a “labelednucleotide”. In some embodiments, the label can be in the form of afluorescent dye attached to any portion of a nucleotide including abase, sugar or any intervening phosphate group or a terminal phosphategroup, i.e., the phosphate group most distal from the sugar.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingany one or any combination of capture primers, reverse primers, fusionprimers, target nucleic acids and/or nucleotides that are non-labeled orattached to at least one label. In some embodiments, the label comprisesa detectable moiety. In some embodiments, the label can generate, orcause to generate, a detectable signal. In some embodiments, thedetectable signal can be generated from a chemical or physical change(e.g., heat, light, electrical, pH, salt concentration, enzymaticactivity, or proximity events). For example, a proximity event caninclude two reporter moieties approaching each other, or associatingwith each other, or binding each other. In some embodiments, thedetectable signal can be detected optically, electrically, chemically,enzymatically, thermally, or via mass spectroscopy or Ramanspectroscopy. In some embodiments, the label can include compounds thatare luminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent or electrochemical. Insome embodiments, the label can include compounds that are fluorophores,chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes.In some embodiments, the label comprises a moiety not typically presentin naturally occurring nucleotides. For example, the label can includefluorescent, luminescent or radioactive moieties.

In some embodiments, the disclosure relates generally to compositions,as well as related, systems, methods, kits and apparatuses, comprisingat least one member of a binding partner. In some embodiments, a bindingpartners includes two molecules, or portions thereof, which have aspecific binding affinity for one another and typically will bind toeach other in preference to binding to other molecules. In someembodiments, binding partners include an “affinity moiety” and a“receptor moiety”. Typically but not necessarily some or all of thestructure of one member of a specific binding pair is complementary tosome or all of the structure possessed by the other member, with the twomembers being able to bind together specifically by way of a bondbetween the complementary structures, optionally by virtue of multiplenon-covalent attractions.

In some embodiments, molecules that function as binding partnersinclude: biotin (and its derivatives) and its binding partners avidin,streptavidin and their derivatives; His-tags which bind nickel, cobaltor copper; cysteine, histidine, or histidine patch which bind Ni-NTA;maltose which binds with maltose binding protein (MBP);lectin-carbohydrate binding partners; calcium-calcium binding protein(CBP); acetylcholine and receptor-acetylcholine; protein A and bindingpartner anti-FLAG antibody; GST and binding partner glutathione; uracilDNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor)protein; antigen or epitope tags which bind to antibody or antibodyfragments, particularly antigens such as digoxigenin, fluorescein,dinitrophenol or bromodeoxyuridine and their respective antibodies;mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound andprotein A; receptor-receptor agonist or receptor antagonist;enzyme-enzyme cofactors; enzyme-enzyme inhibitors; andthyroxine-cortisol. Another binding partner for biotin can be abiotin-binding protein from chicken (Hytonen, et al., BMC StructuralBiology 7:8).

In some embodiments, an avidin moiety can include an avidin protein, aswell as any derivatives, analogs and other non-native forms of avidinthat can bind to biotin moieties. Other forms of avidin moieties includenative and recombinant avidin and streptavidin as well as derivatizedmolecules, e.g. nonglycosylated avidins, N-acyl avidins and truncatedstreptavidins. For example, avidin moiety includes deglycosylated formsof avidin, bacterial streptavidins produced by Streptomyces (e.g.,Streptomyces avidinii), truncated streptavidins, recombinant avidin andstreptavidin as well as to derivatives of native, deglycosylated andrecombinant avidin and of native, recombinant and truncatedstreptavidin, for example, N-acyl avidins, e.g., N-acetyl, N-phthalyland N-succinyl avidin, and the commercial products ExtrAvidin™,Captavidin™, Neutravidin™ and Neutralite Avidin™.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction mixture, which can be used for a nucleic acid synthesis oramplification reaction. The single reaction mixture can include primers(e.g., capture primer, fusion primer, reverse primers, and otheradditional primers), enzymes (e.g., polymerases), accessory proteins(e.g., recombinase, recombinase loading protein, single-stranded bindingprotein, helicase or topoisomerase), nucleotides, divalent cations,binding partners, co-factors and/or buffer. Optionally, the primersinclude any one or any combination of primers attached to a bead (e.g.,immobilized primers) and/or soluble primers. Optionally, the enzymescomprise polymerases which include recombinant, fusion, mutant,heat-stable or heat labile forms. Optionally, the accessory proteinsinclude any one or any combination of a single-stranded binding protein(e.g., SSB or gp32 protein), recombinase (e.g., recA or uvsX),recombinase loading protein (e.g., uvsY protein), helicase (e.g., uvsWprotein), or topoisomerase. Optionally, the nucleotides can includecompounds having structures the same as or similar tonaturally-occurring nucleotides, or nucleotide analogs havingderivatized base, sugar and/or phosphate groups, or labeled ornon-labeled nucleotides. Optionally, the divalent cations includemagnesium, manganese and/or calcium. Optionally, the binding partnersinclude biotin and avidin-like compounds, such as avidin orstreptavidin. Optionally, the buffer comprises a source of ions, such asKCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, or ammonium sulfate.Optionally, the buffer includes Tris, Tricine, HEPES, MOPS, ACES, MES,or inorganic buffers such as phosphate or acetate-based buffers whichcan provide a pH range of about 4-12. Optionally, the buffer includeschelating agents such as EDTA or EGTA. Optionally, the buffer includesdithiothreitol (DTT), glycerol, spermidine, and/or BSA (bovine serumalbumin). Optionally, the buffer includes ATP.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a nucleicacid synthesis or nucleic acid amplification reaction that can beconducted under thermocycling or isothermal conditions, or a combinationof both types of conditions.

In some embodiments thermo-cycling amplification conditions comprise anucleic acid amplification reaction mixture that is subjected to anelevated temperature for a period of time that is sufficient to denatureat least about 30-95% of the double-stranded target nucleic acids, andthen subjected to a lower temperature for a period of time that issufficient to permit hybridization between the single-stranded targetnucleic acids and any of the primers (e.g., capture primer, reverseprimer, or fusion primer).

In some embodiments isothermal amplification conditions comprise anucleic acid amplification reaction mixture that is subjected to atemperature variation which is constrained within a limited range duringat least some portion of the amplification, including for example atemperature variation is within about 20° C., or about 10° C., or about5° C., or about 1-5° C., or about 0.1-1° C., or less than about 0.1° C.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120minutes, or longer.

In some embodiments, an isothermal nucleic acid amplification reactioncan be conducted at about 15-30° C., or about 30-45° C., or about 45-60°C., or about 60-75° C., or about 75-90° C., or about 90-93° C., or about93-99° C.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses, that furtherinclude an enrichment step. In some embodiments, an amplified populationof nucleic acids can include an affinity moiety. For example, inconducting any of the nucleic acid synthesis methods according to thepresent teachings, a reverse primer that is attached to an affinitymoiety (e.g., biotin) can be used to conduct an amplification reactionto produce an amplified population of nucleic acids that are attached tothe affinity moiety. In some embodiments, the enrichment step comprisesforming a enrichment complex by binding the affinity moiety (which isattached to the amplified population of nucleic acids) with apurification bead (e.g., paramagnetic bead) that is attached to areceptor moiety (e.g., streptavidin). An example of purification beadsinclude MyOne™ Beads from Dynabeads, which are paramagnetic beadsattached to streptavidin. In some embodiments, a magnet can be used toseparate/remove the enrichment complex from amplified population ofnucleic acids that lack the affinity moiety.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses that further includeat least one washing step. The washing step can be conducted at any timeduring the method for nucleic acid synthesis. In some embodiments, awashing step can remove excess or unreacted components of the nucleicacid synthesis (e.g., amplification) or enrichment reactions.

In some embodiments, any of the nucleic acid synthesis or amplificationmethods, or enrichment steps, according to the present teachings, can beconducted manually or by automation. In some embodiments, the steps of(1) providing a single reaction mixture, (2) forming any of theamplified populations of nucleic acids, (3) enriching and/or (4)washing, can be conducted manually or by automation. For example, anyreagents for a nucleic acid synthesis (e.g., amplification), enrichmentor washing, can be deposited into, or removed from, a reaction vesselvia manual or automated modes. In some embodiments, reagents for nucleicacid synthesis include, but are not limited to: beads, capture primers,reverse primers, fusion primers, target nucleic acids, enzymes,polymerases, accessory proteins, recombinases, one or more nucleotides,labels, binding partners, divalent cations, and/or co-factors.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a singlereaction vessel containing (i) a first plurality of beads of a firsttype, (ii) a first population of target nucleic acids, (iii) a secondplurality of beads of a second type, and (iv) a second population oftarget nucleic acids.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses), comprise (i) a first plurality of beads of afirst type, (ii) a first population of target nucleic acids, (iii) asecond plurality of beads of a second type, and (iv) a second populationof target nucleic acids, where items (i)-(iv) are deposited into a firstreaction vessel.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses), further comprise (i) a third plurality of beadsof a first type, (ii) a third population of target nucleic acids, (iii)a fourth plurality of beads of a second type, and (iv) a fourthpopulation of target nucleic acids, where items (i)-(iv) are depositedinto a second reaction vessel.

In some embodiments, the compositions (and related methods, systems,kits and apparatuses), comprise (i) at least two different plurality ofbeads of two different type, and (ii) at least two different populationsof target nucleic acids, are deposited into two or more differentreaction vessels.

In some embodiments, the reaction vessel includes a tube (e.g.,Eppendorf™ tube), inner wall of a tube, well, reaction chamber, groove,channel reservoir, flowcell,

In some embodiments, two or more reaction vessels can be two or morereaction chambers arranged in an array. In some embodiments, the arraycan include one or more reaction chambers a solid surface. A reactionchamber can have walls that define width and depth. The dimensions of areaction chamber can be sufficient to permit deposition of reagents orfor conducting reactions. A reaction chamber can have any shapeincluding cylindrical, polygonal or a combination of different shapes.Any wall of a reaction chamber can have a smooth or irregular surface. Areaction chamber can have a bottom with a planar, concave or convexsurface. The bottom and side walls of a reaction chamber can comprisethe same or different material and/or can be coated with a chemicalgroup that can react with a biomolecule such as nucleic acids, proteinsor enzymes.

In some embodiments, the reaction chamber can be one of multiplereaction chambers arranged in a grid or array. An array can include twoor more reaction chambers. Multiple reaction chambers can be arrangedrandomly or in an ordered array. An ordered array can include reactionchambers arranged in a row, or in a two-dimensional grid with rows andcolumns.

An array can include any number of reaction chambers for depositingreagents and conducting numerous individual reactions. For example, anarray can include at least 256 reaction chambers, or at least 256,000,or at least 1-3 million, or at least 3-5 million, or at least 5-7million, or at least 7-9 million, at least 9-11 million, at least 11-13million reaction chambers, or even high density including 13-700 millionreaction chambers or more. Reaction chambers arranged in a grid can havea center-to-center distance between adjacent reaction chambers (e.g.,pitch) of less than about 10 microns, or less than about 5 microns, orless than about 1 microns, or less than about 0.5 microns.

An array can include reaction chambers having any width and depthdimensions. For example, a reaction chamber can have dimensions toaccommodate a single microparticle (e.g., microbead) or multiplemicroparticles. A reaction chamber can hold 0.001-100 picoliters ofaqueous volume.

In some embodiments, at least one reaction vessel (e.g., at least onereaction chamber) can be coupled to one or more sensors or can befabricated above one or more sensors. A reaction chamber that is coupledto a sensor can provide confinement of reagents deposited therein sothat products from a reaction can be detected by the sensor. A sensorcan detect changes in products from any type of reaction, including anynucleic acid reaction such as primer extension, amplification ornucleotide incorporation reactions, within the reaction vessel. A sensorcan detect changes in ions (e.g., hydrogen ions), protons, phosphategroups such as pyrophosphate groups. A sensor can detect at least one byproduct of nucleotide incorporation, including pyrophosphate, hydrogenions, charge transfer, or heat. In some embodiments, at least onereaction chamber can be coupled to one or more field effect transistor(FET), including for example an ion sensitive field effect transistor(ISFET). Examples of an array of reaction chambers coupled to ISFETsensors can be found at U.S. Pat. No. 7,948,015, and U.S. Ser. No.12/002,781, hereby incorporated by reference in their entireties. Otherexamples of sensors that detect byproducts of a nucleotide incorporationreaction can be found, for example, in Pourmand et al, Proc. Natl. Acad.Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,IV-169-172; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006); Esfandyapouret al., U.S. Patent Publication No. 2008/01666727; and Sakurai et al.,Anal. Chem. 64: 1996-1997 (1992).

In some embodiments, the disclosure relates generally to method, andrelated compositions, systems, kits and apparatuses, which furtherinclude a sequencing reaction. In some embodiments, any target nucleicacid that has been amplified according to the present teachings can besequenced.

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forsynthesizing nucleic acids which further comprise sequencing one or morenucleic acids in the first amplified population, or sequencing one ormore nucleic acids in the second amplified population, or sequencing oneor more nucleic acids in both the first and second amplified populationsof target nucleic acids.

In some embodiments, any type of sequencing platform can be employed,including: sequencing by oligonucleotide probe ligation and detection(e.g., SOLiD™ from Life Technologies, WO 2006/084131), probe-anchorligation sequencing (e.g., Complete Genomics™ or Polonator™),sequencing-by-synthesis (e.g., Genetic Analyzer and HiSeq™, fromIIlumina), pyrophosphate sequencing (e.g., Genome Sequencer FLX from 454Life Sciences), ion-sensitive sequencing (e.g., Personal Genome Machine(PGM™) and Ion Proton™ Sequencer, both from Ion Torrent Systems, Inc.),and single molecule sequencing platforms (e.g., HeliScope™ fromHelicos™).

In some embodiments, nucleic acids that have been synthesized, or havebeen amplified, according to the present teachings can be sequenced byany sequencing method, including sequencing-by-synthesis, ion-basedsequencing involving the detection of sequencing byproducts using fieldeffect transistors (e.g., FETs and ISFETs), chemical degradationsequencing, ligation-based sequencing, hybridization sequencing,pyrophosphate detection sequencing, capillary electrophoresis, gelelectrophoresis, next-generation, massively parallel sequencingplatforms, sequencing platforms that detect hydrogen ions or othersequencing by-products, and single molecule sequencing platforms. Insome embodiments, a sequencing reaction can be conducted using at leastone sequencing primer that can hybridize to any portion of thepolynucleotide constructs, including a nucleic acid adaptor or a targetpolynucleotide.

In some embodiments, nucleic acid amplified according to the presentteachings can be sequenced using methods that detect one or morebyproducts of nucleotide incorporation. The detection of polymeraseextension by detecting physicochemical byproducts of the extensionreaction, can include pyrophosphate, hydrogen ion, charge transfer,heat, and the like, as disclosed, for example, in U.S. Pat. No.7,948,015 to Rothberg et al.; and Rothberg et al, U.S. PatentPublication No. 2009/0026082, hereby incorporated by reference in theirentireties. Other examples of methods of detecting polymerase-basedextension can be found, for example, in Pourmand et al, Proc. Natl.Acad. Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,IV-169-172; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006); Esfandyapouret al., U.S. Patent Publication No. 2008/01666727; and Sakurai et al.,Anal. Chem. 64: 1996-1997 (1992).

Reactions involving the generation and detection of ions are widelyperformed. The use of direct ion detection methods to monitor theprogress of such reactions can simplify many current biological assays.For example, template-dependent nucleic acid synthesis by a polymerasecan be monitored by detecting hydrogen ions that are generated asnatural byproducts of nucleotide incorporations catalyzed by thepolymerase. Ion-sensitive sequencing (also referred to as “pH-based” or“ion-based” nucleic acid sequencing) exploits the direct detection ofionic byproducts, such as hydrogen ions, that are produced as abyproduct of nucleotide incorporation. In one exemplary system forion-based sequencing, the nucleic acid to be sequenced can be capturedin a microwell, and nucleotides can be flowed across the well, one at atime, under nucleotide incorporation conditions. The polymeraseincorporates the appropriate nucleotide into the growing strand, and thehydrogen ion that is released can change the pH in the solution, whichcan be detected by an ion sensor that is coupled with the well. Thistechnique does not require labeling of the nucleotides or expensiveoptical components, and allows for far more rapid completion ofsequencing runs. Examples of such ion-based nucleic acid sequencingmethods and platforms include the Ion Torrent PGM™ or Proton™ sequencer(Ion Torrent™ Systems, Life Technologies Corporation).

In some embodiments, target polynucleotides produced using the methods,systems and kits of the present teachings can be used as a substrate fora biological or chemical reaction that is detected and/or monitored by asensor including a field-effect transistor (FET). In various embodimentsthe FET is a chemFET or an ISFET. A “chemFET” or chemical field-effecttransistor, is a type of field effect transistor that acts as a chemicalsensor. It is the structural analog of a MOSFET transistor, where thecharge on the gate electrode is applied by a chemical process. An“ISFET” or ion-sensitive field-effect transistor, is used for measuringion concentrations in solution; when the ion concentration (such as H+)changes, the current through the transistor will change accordingly. Adetailed theory of operation of an ISFET is given in “Thirty years ofISFETOLOGY: what happened in the past 30 years and what may happen inthe next 30 years,” P. Bergveld, Sens. Actuators, 88 (2003), pp. 1-20.

In some embodiments, the FET may be a FET array. As used herein, an“array” is a planar arrangement of elements such as sensors or wells.The array may be one or two dimensional. A one dimensional array can bean array having one column (or row) of elements in the first dimensionand a plurality of columns (or rows) in the second dimension. The numberof columns (or rows) in the first and second dimensions may or may notbe the same. The FET or array can comprise 102, 103, 104, 105, 106, 107or more FETs.

In some embodiments, one or more microfluidic structures can befabricated above the FET sensor array to provide for containment and/orconfinement of a biological or chemical reaction. For example, in oneimplementation, the microfluidic structure(s) can be configured as oneor more wells (or microwells, or reaction chambers, or reaction wells,as the terms are used interchangeably herein) disposed above one or moresensors of the array, such that the one or more sensors over which agiven well is disposed detect and measure analyte presence, level,and/or concentration in the given well. In some embodiments, there canbe a 1:1 correspondence of FET sensors and reaction wells.

Microwells or reaction chambers are typically hollows or wells havingwell-defined shapes and volumes which can be manufactured into asubstrate and can be fabricated using conventional microfabricationtechniques, e.g. as disclosed in the following references: Doering andNishi, Editors, Handbook of Semiconductor Manufacturing Technology,Second Edition (CRC Press, 2007); Saliterman, Fundamentals of BioMEMSand Medical Microdevices (SPIE Publications, 2006); Elwenspoek et al,Silicon Micromachining (Cambridge University Press, 2004); and the like.Examples of configurations (e.g. spacing, shape and volumes) ofmicrowells or reaction chambers are disclosed in Rothberg et al, U.S.patent publication 2009/0127589; Rothberg et al, U.K. patent applicationGB24611127.

In some embodiments, the biological or chemical reaction can beperformed in a solution or a reaction chamber that is in contact with,operatively coupled, or capacitively coupled to a FET such as a chemFETor an ISFET. The FET (or chemFET or ISFET) and/or reaction chamber canbe an array of FETs or reaction chambers, respectively.

In some embodiments, a biological or chemical reaction can be carriedout in a two-dimensional array of reaction chambers, wherein eachreaction chamber can be coupled to a FET, and each reaction chamber isno greater than 10 μm³ (i.e., 1 pL) in volume. In some embodiments eachreaction chamber is no greater than 0.34 pL, 0.096 pL or even 0.012 pLin volume. A reaction chamber can optionally be no greater than 2, 5,10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns incross-sectional area at the top. Preferably, the array has at least 10²,10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or more reaction chambers. In someembodiments, at least one of the reaction chambers is operativelycoupled to at least one of the FETs.

FET arrays as used in various embodiments according to the disclosurecan be fabricated according to conventional CMOS fabricationstechniques, as well as modified CMOS fabrication techniques and othersemiconductor fabrication techniques beyond those conventionallyemployed in CMOS fabrication. Additionally, various lithographytechniques can be employed as part of an array fabrication process.

Exemplary FET arrays suitable for use in the disclosed methods, as wellas microwells and attendant fluidics, and methods for manufacturingthem, are disclosed, for example, in U.S. Patent Publication No.20100301398; U.S. Patent Publication No. 20100300895; U.S. PatentPublication No. 20100300559; U.S. Patent Publication No. 20100197507,U.S. Patent Publication No. 20100137143; U.S. Patent Publication No.20090127589; and U.S. Patent Publication No. 20090026082, which areincorporated by reference in their entireties.

In one aspect, the disclosed methods, compositions, systems, apparatusesand kits can be used for carrying out label-free nucleic acidsequencing, and in particular, ion-based nucleic acid sequencing. Theconcept of label-free detection of nucleotide incorporation has beendescribed in the literature, including the following references that areincorporated by reference: Rothberg et al, U.S. patent publication2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129: 79-86(2008); and Pourmand et al, Proc. Natl. Acad. Sci., 103: 6466-6470(2006). Briefly, in nucleic acid sequencing applications, nucleotideincorporations are determined by measuring natural byproducts ofpolymerase-catalyzed extension reactions, including hydrogen ions,polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase).Examples of such ion-based nucleic acid sequencing methods and platformsinclude the Ion Torrent PGM™ or Proton™ sequencer (Ion Torrent™ Systems,Life Technologies Corporation).

In some embodiments, the disclosure relates generally to methods forsequencing nucleic acids that have been amplified by the teachingsprovided herein. In one exemplary embodiment, the disclosure relatesgenerally to a method for obtaining sequence information frompolynucleotides, comprising: (a) amplifying nucleic acids; and (b)performing template-dependent nucleic acid synthesis using at least oneof the amplified nucleic acids produced during step (a) as a template.The amplifying can optionally be performed according to any of theamplification methods described herein.

In some embodiments, the template-dependent synthesis includesincorporating one or more nucleotides in a template-dependent fashioninto a newly synthesized nucleic acid strand.

Optionally, the methods can further include producing one or more ionicbyproducts of such nucleotide incorporation.

In some embodiments, the methods can further include detecting theincorporation of the one or more nucleotides into the sequencing primer.Optionally, the detecting can include detecting the release of hydrogenions.

In another embodiment, the disclosure relates generally to a method forsequencing a nucleic acid, comprising: (a) amplifying nucleic acidsaccording to the methods disclosed herein; (b) disposing the amplifiednucleic acids into a plurality of reaction chambers, wherein one or moreof the reaction chambers are in contact with a field effect transistor(FET). Optionally, the method further includes contacting amplifiednucleic acids which are disposed into one of the reaction chambers, witha polymerase thereby synthesizing a new nucleic acid strand bysequentially incorporating one or more nucleotides into a nucleic acidmolecule. Optionally, the method further includes generating one or morehydrogen ions as a byproduct of such nucleotide incorporation.Optionally, the method further includes detecting the incorporation ofthe one or more nucleotides by detecting the generation of the one ormore hydrogen ions using the FET.

In some embodiments, the detecting includes detecting a change involtage and/or current at the at least one FET within the array inresponse to the generation of the one or more hydrogen ions.

In some embodiments, the FET can be selected from the group consistingof: ion-sensitive FET (isFET) and chemically-sensitive FET (chemFET).

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ orProton™ sequencer (Life Technologies), which is an ion-based sequencingsystem that sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencer detects the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencer caninclude a plurality of nucleic acid templates to be sequenced, eachtemplate disposed within a respective sequencing reaction well in anarray. The wells of the array can each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor can provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types can be flowedserially into the reaction chamber, and can be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation can be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions can be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer multiple nucleotide flows into the reaction chamberalong with incorporation monitoring across a multiplicity of wells orreaction chambers can permit the instrument to resolve the sequence ofmany nucleic acid templates simultaneously. Further details regardingthe compositions, design and operation of the Ion Torrent PGM™ orProton™ sequencer can be found, for example, in U.S. patent applicationSer. No. 12/002,781, now published as U.S. Patent Publication No.2009/0026082; U.S. patent application Ser. No. 12/474,897, now publishedas U.S. Patent Publication No. 2010/0137143; and U.S. patent applicationSer. No. 12/492,844, now published as U.S. Patent Publication No.2010/0282617, all of which applications are incorporated by referenceherein in their entireties.

EXAMPLES

Embodiments of the present teachings can be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, (a)providing a plurality of different populations of target nucleic acids,and a plurality of different types of discrete supports, and (b) formingdifferent amplified populations of the target nucleic acids that areattached to the different types of discrete supports. Four differenthuman genomic DNA fragment libraries were prepared. The librariescontained a 200 bp insert, and included an L1 (SEQ ID NOS:11 and 12), L2(SEQ ID NOS:13 and 14), L3 (SEQ ID NOS: 15 and 16) or L4 double-strandedadaptor (SEQ ID NOS: 17 and 18) joined at one end and a universaladaptor A joined at the other end. Four different types of beads wereprepared, each type of bead covalently attached with a plurality of onetype of capture primers, AV1, AV2, AV3, or AV4 (SEQ ID NOS:1-4,respectively). A 1.2 mL aqueous mixture was prepared that containedabout 250 million molecules of each of the four different humanlibraries, and about 1.5 billion beads of each of the four types, andfour different types of fusion primers (AV1_L1, AV2_L2, AV3_L3, andAV4_L4) (SEQ ID NOS:23-26, respectively), and universal reverse Aprimers. A water-in-oil emulsion was prepared with the 1.2 mL mixture.

A control 1.2 mL emPCR reaction mixture was prepared. The control emPCRmixture contained about 250 million molecules of one type of humanlibrary (which included a 200 bp insert) with an AV1 adapter (SEQ IDNOS:11 and 12) joined to one end, and the universal adaptor A joined atthe other end, and about 6 billion of one type of bead (B beads), andB_AV1 fusion primers and universal reverse A primers.

The emPCR procedure, using thermocycling, was conducted according tomanufacturer's instructions contained in the user manual Ion PI™Template OT2 200 Kit v3 (publication No. MAN0009133).

The templated beads from each reaction were sequenced separately on anIon Torrent™ PI chip. The sequencing data as aligned to the humangenome, and the number of duplicate sequences in each set of alignmentswas determined. The reaction containing four bead types produced aduplicate rate of 9.4% whereas the control reaction with only one beadtype produced a duplicate rate of 26.5%. Other metrics such as yield oftemplated beads, polyclonal percentage, and number of alignments weresimilar between the two conditions (see Table 1).

TABLE 1 number of templated percent duplicate bead types bead yieldpolyclonal alignments rate 1 584,000,000 32% 77,220,582 26.5% 4554,000,000 30% 84,950,739  9.4%

Example 2

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, (a)providing a plurality of different populations of target nucleic acids,and a plurality of different types of discrete supports, and (b) formingdifferent amplified populations of the target nucleic acids that areattached to the different types of discrete supports. Four differenthuman genomic DNA fragment libraries were prepared. The librariescontained a 200 bp insert, and included an L1 (SEQ ID NOS:11 and 12), L2(SEQ ID NOS:13 and 14), L3 (SEQ ID NOS:15 and 16) or L4 double-strandedadaptor (SEQ ID NOS:17 and 18) joined at one end and a universal adaptorA joined at the other end. Four different types of beads were prepared,each type of bead covalently attached with a plurality of one type ofcapture primers, AV1, AV2, AV3, or AV4 (SEQ ID NOS:1-4, respectively). A2.4 mL aqueous mixture was prepared that contained about 125 millionmolecules of each of the four different human libraries, and about 1.5billion beads of each of the four types, and four different types offusion primers (AV1_L1, AV2_L2, AV3_L3, and AV4_L4) (SEQ ID NOS:23-26,respectively) and universal reverse A primers. A water-in-oil emulsionwas prepared with the 2.4 mL mixture.

A control 2.4 mL emPCR reaction mixture was prepared. The control emPCRmixture contained about 125 million molecules of one type of humanlibrary (which included a 200 bp insert) with an AV1 adapter (SEQ IDNOS:11 and 12) joined to one end, and the universal adaptor A joined atthe other end, about 6 billion of one type of bead (B beads), and B_AV1fusion primers and reverse universal A primers.

The emPCR procedure, using thermocycling, was conducted according tomanufacturer's instructions contained in the user manual Ion PI™Template OT2 200 Kit v3 (publication No. MAN0009133).

Typically, this emPCR reaction is conducted using 500 million moleculesper bead type, which yields approximately 30% polyclonality. In thisreaction, only 125 million library molecules per bead type were includedin the emPCR reaction mixture.

The templated beads from each reaction were sequenced separately,aligned to the human genome, and the number of duplicate sequences ineach set of alignments was determined. Both reactions produced a lownumber of polyclonal beads (19% and 21%, see Table 2). However, thereaction containing four bead types produced a duplicate rate of 12%whereas the control reaction with only one bead type produced aduplicate rate of 27.7%. Other metrics such as yield of templated beadsand number of alignments were similar between the two conditions (seeTable 2).

TABLE 2 number of templated percent duplicate bead types bead yieldpolyclonal alignments rate 1 161,000,000 21% 64,993,033 27.7% 4134,000,000 19% 74,452,984 12.0%

Example 3

In some embodiments, the disclosure relates generally to methods, aswell as related, systems, compositions, kits and apparatuses, forpreparing an enriched sample containing templated beads attached to asubstantially monoclonal population of one of target nucleic acids. Insome embodiments, the enrichment steps are performed manually or byautomation. For example, automated enrichment steps can be conductedusing an Ion Torrent One Touch apparatus and kit (e.g., Ion PGM™Template OT2 200 Kit, from Ion Torrent™ Systems, Life TechnologiesCorporation). Alternatively, manual enrichment steps can be conductedusing the reagents and solutions from an Ion PGM™ Template OT2 200 Kit,without the OT2™ apparatus. Prepare fresh Melt-Off solution by mixingtogether 280 uL Tween Solution (from the Ion PGM™ Template OT2 200 Kit)and 40 uL of 1 M NaOH. Prepare fresh 3 mM SDS. Remove the RecoverySolution from the Recovery tubes, leaving approximately 100 uL in thetubes. Pool the remaining Recovery Solution into a 1.7 mL tube. Rinsethe Recovery Tubes with approximately 100 uL of water, and add to the1.7 mL tube. Bring the volume of the 1.7 mL tube to 1000 mL with water.Optionally, remove an aliquot for Guava. Split into two 1.7 mL tubes,and add 500 uL of One Touch Wash Solution to each 1.7 mL tube. Vortexthe MyOne beads for about 30 seconds. Add about 100 uL of unwashedOneTouch™ beads to the 1.7 mL tubes. Vortex the 1.7 mL tubes for about30 seconds. Incubate at room temperature for about 2 minutes. Place thetubes on a magnet (e.g., DynaMag™ from Life Technologies) for separationfor about 2 minutes. Remove the supernatant and discard. Add 200 uL ofthe Melt-Off solution to the tubes. Vortex the tubes for about 30seconds and centrifuge for about 2 seconds. Incubate at room temperaturefor about 2 minutes. Place the tubes on the magnet for about 2 minutes.Remove the supernatant (contains an enriched population of templatedbeads) and add to a fresh 1.7 mL tube (enrichment tube). Bring thevolume of the enrichment tube to 1000 uL with water. Centrifuge forabout 8 minutes at about 15,500 xg to pellet the enriched population oftemplated beads. Remove the supernatant, leaving about 20 uL, anddispose the supernatant. Add about 180 uL of nuclease-free water to four0.2 mL tubes. Remove about 1 uL of the enriched sample for Guavaanalysis. Split the enriched sample into the four 0.2 mL tubes, andbring the volume in each tube to 200 uL with water. Centrifuge the 0.2mL tubes for about 5 minutes at about 15,500 xg to pellet the enrichedtemplated beads. Remove the supernatant and dispose, leaving theenriched templated beads in the pellet in about 10 uL liquid. To each0.2 mL tube, add 20 uL of Annealing Buffer and 20 uL of SequencingBuffer and mix (both buffers from an Ion Torrent Sequencing kit fromLife Technologies). Anneal the sequencing primers by incubating in athermocycler (using the heated lid option) at 95 degrees for 120seconds, and 37 degrees for 120 seconds. After the annealing step, poolthe samples and add 40 uL loading buffer. Load several PGM or PI IonTorrent sequencing chips, and conduct the sequencing steps according tothe manufacture instructions (e.g., Ion PGM Sequencing 200 Kit v2,catalog No. 4482006, or Ion Pt Sequencing 200 Kit v3, catalog No.4488315).

Example 4

Another example for the preparation of an enriched sample containingtemplated particles (e.g., beads) in accordance with the presentdisclosure may be performed using the following protocol.

Library Preparation:

For library preparation, follow the preparation workflow instructions inthe Ion Xpress™ Plus gDNA Fragment Library Preparation User Guide(MAN0009847 Rev C.0) for product information, and required materials andequipment, and in Ion Xpress™ Plus gDNA Fragment Library PreparationUser Guide (MAN0009847 Rev C.0) for gDNA fragmentation. Proceed with thepreparation using the above instructions up until the adapter ligationand nick-repair procedure of Chapter 4, “Ligate adapters, nick-repair,and purify the ligated DNA”.

Perform ligation and nick-repair as follows. In a 0.2-mL tube (e.g., PCRtube), combine the reagents as indicated in the appropriate table fornon-barcoded or barcoded libraries, and mix well by pipetting up anddown. For barcoded libraries, use the table below for the reaction setupup:

Reaction Setup for Barcoded Libraries Volume by Input gDNA Component50-100 ng 1 μg DNA ~25 μL ~25 μL 10X Ligase Buffer 10 μL 10 μL MosaicLibrary Adaptors 2 μL 10 μL Ion Xpress Barcode X 2 μL 10 μL dNTP Mix 2μL 2 μL Nuclease-free Water 49 μL 31 μL DNA Ligase 2 μL 4 μL Nick RepairPolymerase 8 μL 8 μL Total 100 μL 100 μL

Once the tube reaction mixture outlined above has been generated, andmixed via pipetting, place the tube in a thermal cycler and thermalcycle the tube as follows:

Stage Temperature Time Hold 25° C. 15 min Hold 72° C.  5 min Hold  4° C.HoldThen, transfer the entire reaction mixture to a 1.5-mL tube (e.g.,Eppendorf LoBind® Tube) for the next cleanup step. For cleanup, followthe instructions in the Ion Xpress™ Plus gDNA Fragment LibraryPreparation (MAN0009847 Rev C.0) at Chapter 4, “Purify theadapter-ligated and nick-repaired DNA” and Chapter 5, “Size-select theunamplified library-Option 2: Size-Select the library with Pippin Prep™instrument”.

For 220-base-read libraries, from the cassette type drop-down menu usingthe Pippin Prep™ instrument, choose 2% Marker B No Overflow Detection.Select the Tight collection mode for each lane and then define the BPTarget setting for each of 1-4 lanes used.

Sequencing System Library Size BP Target Setting Proton 220-base reads310 bpDefine lanes 1-4 as sample lanes and 5 as the ladder lane by entering“5” in the reference lane box and selecting the Apply Reference to allLanes button. Ensure that the “Ref Lane” value for each lane is 5. Setthe run time for 1.5 hours. And then proceed to “LOAD the Sample: For100-300-base read libraries”. Once completed, quantify the library usingthe Ion Library Quantitation Kit (Cat. No. 4468802).

Next, perform a templating reaction using an OT2 PSP4. For thetemplating reaction, refer to the Ion PI™ Template OT2 200 Kit v3 UserGuide (MAN0009133 Revision B.0) for production information, and requiredmaterials and equipment.

Dispense 150 μL Ion PI™ OT2 Breaking Solution into each of two tubes(e.g., Recovery Tubes). Add 4 μL of the 1 mM AV1-4 Mosaic Oligo Pool tothe 150 Breaking Solution in each Recovery Tube. Install the tubes andthe Ion OneTouch™ Recovery Router, then close the centrifuge lid. Followthe procedure outlined in the Ion PI™ Template OT2 200 Kit v3 User Guide(MAN0009133Revision B.0) for installation of the Ion OneTouch™ 2Amplification Plate, disposable injector, and reagents.

Next, prepare the amplification solution as follows. Vortex the AV1-4Ion Sphere™ Particle Mix at maximum speed for 1 minute, centrifuge for 2seconds, pipet up and down to mix; then immediately proceed to the nextstep. In a 2.5-mL Reaction Tube at 15° C. to 30° C., add the followingcomponents in the designated order. Add each component, then pipette theamplification solution up and down to mix:

Order Reagent Volume 1 Nuclease-Free Water 160 μL 3 1.2x Taq LR1Amplification Mix 2000 μL 4 30x Taq LR1 Enzyme 120 μL 5 Mosaic AV1-4 IonSphere Mix 100 μL 6 Mosaic Library (100 pM) 28.8 μL 7 Fusion Primer Pool(100 uM) 4.8 μL Total 2400 μLVortex the complete amplification solution prepared at maximum speed for5 seconds. Proceed immediately to follow the instructions found in “Filland install the Ion PI™ Plus Reaction Filter Assembly on the IonOneTouch™ 2 Instrument” described in the Ion PI™ Template OT2 200 Kit v3User Guide (MAN0009133Revision B.0).

After amplification, the template-positive particles (Ion PI™ IonSphere™ Particles) are recovered. Reference is made to the production,information, materials and equipment outlined in the Ion PI™ TemplateOT2 200 Kit v3 User Guide (MAN0009133 Revision B.0). At the end of theamplification run, follow the screen prompts to centrifuge the sample.If reaction tubes are removed at the end of the run before the IonOneTouch™ 2 Instrument has spun the sample or has not processed thesample after 15 minutes, centrifuge the sample on the instrument asfollows:

-   -   a. On the home screen of the instrument, touch Open Lid, wait        until the lid clicks open, then insert the two filled Ion        OneTouch™ Recovery Tubes from the run in the centrifuge rotor.        Close the lid until it locks.    -   b. Touch Options, then touch Final Spin (see figure below), then        follow the screen prompts (touch Next on the next 2 screens)        until the centrifugation begins. Centrifugation of the samples        takes 10 minutes.

Immediately after the centrifuge has stopped, on the instrument display,touch Open Lid, wait until the lid clicks open, then remove and discardthe Ion OneTouch™ Recovery Router. Remove both Ion OneTouch™ RecoveryTubes from the instrument, and put the two Recovery Tubes in a tuberack. Some cloudiness in the tube may be observable, which is normal.

Without any intervening storage of the recovered, template-positiveparticles at a reduced temperature, proceed immediately to washing thetemplate-positive particles. Label a new 1.5 mL tube (e.g., 1.5-mLEppendorf LoBind® Tube) for the template-positive particles. Use apipette to remove all but about 100 μL of Ion OneTouch™ RecoverySolution from each tube (e.g., Ion OneTouch™ Recovery Tube). Withdrawthe supernatant from the surface and on the opposite side from thepellet. Do not disturb the pellet of template-positive particles.Resuspend the template-positive particles in the remaining RecoverySolution in each tube by pipetting the suspension up and down. Transferthe suspension from each Recovery Tube to the new labeled tube (e.g.,1.5-mL tube Eppendorf LoBind® Tube). Add 100 μL of Nuclease-free Waterto each of the Recovery Tubes, then pipette each aliquot in the tube upand down to mix and recover residual particles. Transfer the 100-μLaliquot from each Recovery Tube to the new labeled tube (e.g., 1.5-mLEppendorf LoBind® Tube) to combine the aliquots.

Bring the volume of the suspension to 1 mL with Nuclease-free Water. Thetemplate-positive particles can be stored at 2° C. to 8° C. for up to 3days. Do not store the recovered template-positive particles in IonOneTouch™ Recovery Solution. After storage for up to 3 days, vortex thetube of template-positive particles for 30 seconds to completelyresuspend the particles, and then centrifuge the tube for 2 seconds.Take 2 uL for Qubit assay. Split the sample into total of 2-1.5 mL tubesand add 500 uL of wash solution (e.g., Ion OneTouch™ Wash Solution).Vortex e Dynabeads® MyOne™ Streptavidin Cl Beads for 30 seconds and add100 uL unwashed e Dynabeads® MyOne™ Streptavidin Cl Beads to each tube.Vortex the tubes containing the template-positive particles and eDynabeads for 30 Seconds and incubate at room temperature for 2 minutes.Place the tubes on a magnet (e.g., DynaMag™ from Life Technologies) andallow particles to pellet to wall of tubes. Remove supernatant withoutdisturbing the pellet and add 1 mL Ion PI™ ES Wash Solution (W). Removefrom the magnet and vortex for 30 seconds and return to the magnet forseparation. Remove supernatant and dispense 200 uL freshly made melt offsolution and vortex for 30 sec, quick spin and incubate at roomtemperature for 2 minutes. Again, place the tubes on the magnet forseparation.

At this point, the enriched particles are now in supernatant, and thesupernatant should not be discarded. Transfer and pool both 1.7 mLsupernatants into a new, labelled 1.7 mL tube. Bring the volume of thecontents of the tube to 1 mL with Nuclease free water and centrifuge for8 minutes at 15,500×g. Bring the volume of the tube down to about 20 uLand dispense about 180 uL Nuclease free water bringing the volume to 200uL. Pipette up and down to resuspend beads and take 2 uL for Qubit.Split samples equally across four (4) 0.2 mL tubes (e.g., PCR tubes) andbring volume to 200 ul with Nuclease free water.

At this point, sequencing can be performed as outlined in the Ion PI™Sequencing 200 Kit v3 (MAN0009136 Revision B.0).

Example 5

The protocol described in Example 4 (above) was used to prepare 2batches of templated beads, and to compare bead clumping by conducting anucleic acid synthesis reaction, in a single reaction vessel with anemulsion, using four different types of beads. In a first batch, thebead templating reaction employed a set of four different beads/captureprimers and their cognate adaptors, fusion primers, and reverse primers(FIG. 4A). The first batch included capture primers AV1, AV2, AV3 andAV4 (SEQ ID NOS:1-4, respectively); double-stranded adaptors L1 (SEQ IDNOS:11 and 12), L2 (SEQ ID NOS:13 and 14), L3 (SEQ ID NOS:15 and 16) andL4 (SEQ ID NOS:17 and 18); and fusion primers AV1_L1, AV2_L2, AV3_L3,and AV4_L4) (SEQ ID NOS:23-26, respectively). In a second batch, thebead templating reaction employed an improved set of four differentbeads/capture primers and their cognate adaptors, fusion primers, andreverse primers, that were designed to decrease primer-dimer formation(FIG. 4B). The second batch include capture primers AV1, AV3, AV5 andAV6 (SEQ ID NOS:1, 3, 5 and 6, respectively); double-stranded adaptorsL1 (SEQ ID NOS:11 and 12), L3 (SEQ ID NOS:15 and 16), L5 (SEQ ID NOS:19and 20) and L6 (SEQ ID NOS:21 and 22); fusion primers AV1_(—)11, AV3_L3,AV5_L5 and AV6_L6 (SEQ ID NOS:23, 25, 27 and 28, respectively). Theprofile of templated beads was monitored using a fluorescently-labeledprobe. The data in FIGS. 4A and B demonstrates that the amount of beadclumping is reduced when conducting the bead templating reaction withthe improved set of primers.

Example 6

The protocol described in Example 4 (above) was used to prepare 2batches of templated beads, and to compare bead clumping by conducting anucleic acid synthesis reaction, in a single reaction vessel with anemulsion, using four different types of beads. In a first batch, thebead templating reaction employed a set of four different beads/captureprimers and their cognate adaptors, fusion primers, and reverse primers(FIGS. 5A and B). The first batch included capture primers AV1, AV2, AV3and AV4 (SEQ ID NOS:1-4, respectively); double-stranded adaptors L1 (SEQID NOS:11 and 12), L2 (SEQ ID NOS:13 and 14), L3 (SEQ ID NOS:15 and 16)and L4 (SEQ ID NOS:17 and 18); and fusion primers AV1_L1, AV2_L2,AV3_L3, and AV4_L4) (SEQ ID NOS:23-26, respectively). In a second batch,the bead templating reaction employed an improved set of four differentbeads/capture primers and their cognate adaptors, fusion primers, andreverse primers, that were designed to decrease primer-dimer formation(FIGS. 5C and D). The second batch include capture primers AV1, AV3, AV5and AV6 (SEQ ID NOS:1, 3, 5 and 6, respectively); double-strandedadaptors L1 (SEQ ID NOS:11 and 12), L3 (SEQ ID NOS:15 and 16), L5 (SEQID NOS:19 and 20) and L6 (SEQ ID NOS:21 and 22); fusion primersAV1_(—)11, AV3_L3, AV5_L5 and AV6_L6 (SEQ ID NOS:23, 25, 27 and 28,respectively). The profile of templated beads was monitored using afluorescently-labeled probe. FIGS. 5A and B show pre-enriched andenriched templated beads, respectively, that were prepared using theoriginal set of primers. FIGS. 5C and D show pre-enriched and enrichedtemplated beads, respectively, that were prepared using the improved setof primers. The circled regions in FIGS. 5A-D indicate the poorlytemplated and clumped beads. The data in FIGS. 5A-D demonstrates thatthe amount of bead clumping is reduced when conducting the beadtemplating reaction with the improved set of primers.

Example 7

The protocol described in Example 4 (above) was used to prepare 4batches of templated beads, by conducting a nucleic acid synthesisreaction, in a single reaction vessel with an emulsion, using fourdifferent types of beads and a library containing 220 bp targetpolynucleotides inserts from a human DNA sample (NA12878). Two batchesof the resulting templated beads were combined and then loaded onto sixIon Torrent™ Proton I™ chips, and sequenced to achieve approximately 30Xcoverage (see Table 3 below). For example, each of the bead templatingreactions contained four types of beads (AV1+AV3+AV5+AV6).

TABLE 3 V2 2X OT V2 2X OT combined Set 1 combined Set 2 Total alignedreads (M) 547 M 503 M Total aligned bases (G) 104 G 95.3 G Coverage 33.9X 30.8 X Polyclonality 26% 25% Low Quality  6%  9% AQ17 mean read length182 180 Duplicate rate (start + end) 13% 12%

Example 8

The protocol described in Example 4 (above) was used to preparetemplated beads, and to compare duplicate bead formation by conducting anucleic acid synthesis reaction, in a single reaction vessel with anemulsion, using one, two, three and four different types of beads, andthe resulting templated beads were loaded onto four Ion Torrent™ ProtonI™ chips, and sequenced.

For example, the bead templating reactions contained one type of bead(P1), two types of beads (AV1+AV3), three types of beads (AV1+AV3+AV6),and four types of beads (AV1+AV3+AV5+AV6). The data presented in Table 4below and in FIG. 6 shows the combined duplicate rate per number ofdifferent bead types loaded onto 4 Ion Proton I™ chips per singleemulsion. The bead templating reaction contained 18 billion beads and350 million each library molecules.

TABLE 4 Metric (merged) AV1 + 3 + 5 + 6 AV1 + 3 + 6 AV1 + 3 P1 TotalReads (M) 359 375 383 331 Total Aligned 67.1 70.9 74.3 56.4 Bases (G)Duplicates   17% 25% 30% 39% (start pos) Duplicates 13.5% 21% 28% 36%(start pos + insert end flow)

The data in Table 4 and FIG. 6 demonstrate that the duplicate beadpercent decreases from approximately 36% for one type of bead, toapproximately 13% for four different types of beads.

Example 9

The protocol described in Example 4 (above) was used to preparetemplated beads, and to compare duplicate bead formation by conducting anucleic acid synthesis reaction, in a single reaction vessel with anemulsion, using one, two, three and four different types of beads, andthe resulting templated beads were loaded onto one, three, or four IonTorrent™ Proton I™ chips, and sequenced (see FIG. 7). For example, thebead templating reactions contained one type of bead (P1), two types ofbeads (AV1+AV3), three types of beads (AV1+AV3+AV6), and four types ofbeads (AV1+AV3+AV5+AV6).

The graph in FIG. 7 shows duplicate bead production for bead templatingreactions containing one, two, three or four different types of beads,which were loaded onto 4 Ion Proton I™ chips (♦), which yielded about370 million reads. The (♦) represents data for duplicate bead rate(start+end) and is similar to the calculated linear duplicate rate.

The graph in FIG. 7 also shows duplicate bead production for beadtemplating reactions containing one, two, three or four different typesof beads, which were loaded onto 3 Ion Proton I™ chips (▪), whichyielded about 275 million reads. The (▪) represents data for duplicatebead rate (start+end) and is similar to the calculated linear duplicaterate.

The graph in FIG. 7 also shows duplicate bead production for beadtemplating reactions containing one, two, three or four different typesof beads, which were loaded onto 1 Ion Proton I™ chip (▴), which yieldedabout 80 million reads. The (▴) represents data for duplicate bead rate(start+end) and is similar to the calculated linear duplicate rate.

The sequences of the capture primers, adaptors and fusion primers, thatwere used to conduct the experiments described in Examples 1-9 above,are listed below.

Bead capture primers: AV1: (SEQ ID NO: 1)5′-GCACACATTCAGAGTCAGCAGCTCAGCATCAT AV2: (SEQ ID NO: 2)5′-TTAGGAGATGTTCATGCAGACTCACGATCAGT AV3: (SEQ ID NO: 3)5′-AGTCACTTATCATCGGTGGTACGCAGCTCATT AV4: (SEQ ID NO: 4)5′-ATTCGAGCTGTTCATCTGTATCTTGCGCTACCAA AV5: (SEQ ID NO: 5)5′-GAATCTGTTCTCACTATTCACGCTGGAGGAGT AV6: (SEQ ID NO: 6)5′-GTAACTCGATCAGGTCACACGACCGTTCTCAGCAT AV7: (SEQ ID NO: 7)5′-GATTTCGCAGCTAACCTTGGTGGAAGCTCTCAT AV8: (SEQ ID NO: 8)5′-CTTGAACTACACCACTCTGATGTGCCAGTCTA AV9: (SEQ ID NO: 9)5′-TATCGAGCTAGTGCGTGCTATCAGAACCTATCAGT AV10: (SEQ ID NO: 10)5′-ACGTCGACACTAGCTACCTGTCAGCTACGTGTALibrary adaptors (comprising top and bottomstrands, and having phosphorothiolate,phosphorothioate, and/or phosphoramidate linkages (*)): ML1a:(SEQ ID NO: 11) 5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG AAGCTCTCATGATML1b: (SEQ ID NO: 12) 5′-ATCATGAGAGCTTCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T ML2a: (SEQ ID NO: 13)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG TGCCAGTCTAGAT ML2b:(SEQ ID NO: 14) 5′-ATCTAGACTGGCACACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T ML3a: (SEQ ID NO: 15)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG ACCTATCAGTGAT ML3b:(SEQ ID NO: 16) 5′-ATCACTGATAGGTCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T ML4a: (SEQ ID NO: 17)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG GCTACGTGTAGAT ML4b:(SEQ ID NO: 18) 5′-ATCTACACGTAGCCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T ML5a: (SEQ ID NO: 19)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG TGCCAGTCTAGAT ML5b:(SEQ ID NO: 20) 5′-ATCTAGACTGGCACACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T ML6a: (SEQ ID NO: 21)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTG GCTACGTGTAGAT ML6b:(SEQ ID NO: 22) 5′-ATCTACACGTAGCCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T P1 (a) adaptor: (SEQ ID NO: 30)5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGC AGTCGGTGAT P1 (b) adaptor:(SEQ ID NO: 31) 5′-ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCG TAGTGG*T*TFusion primers: AV1/ML1: (SEQ ID NO: 23)5′-GCACACATTCAGAGTCAGCAGCTCAGCATCATGCAGTCG GTGAAGCTCTCAT AV2/ML2:(SEQ ID NO: 24) 5′-TTAGGAGATGTTCATGCAGACTCACGATCAGTGCAGTCG GTGTGCCAGTCTAAV3/ML3: (SEQ ID NO: 25) 5′-AGTCACTTATCATCGGTGGTACGCAGCTCATTGCAGTCGGTGACCTATCAGT AV4/ML4: (SEQ ID NO: 26)5′-ATTCGAGCTGTTCATCTGTATCTTGCGCTACCAAGCAGTCG GTGGCTACGTGTA AV5/ML5:(SEQ ID NO: 27) 5′-GAATCTGTTCTCACTATTCACGCTGGAGGAGTGCAGTCG GTGTGCCAGTCTAAV6/ML6: (SEQ ID NO: 28) 5′-GTAACTCGATCAGGTCACACGACCGTTCTCAGCATGCAGTCGGTGGCTACGTGTA B/P1: (SEQ ID NO: 29)5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCCTCTCTAT GGGCAGTCGGTGAT

What is claimed:
 1. A method of nucleic acid synthesis, comprising: a)providing a single reaction mixture containing a plurality of beads of afirst type, the beads of the first type including first capture primers;a plurality of beads of a second type, the beads of the second typeincluding second capture primers, wherein the first and second captureprimers are different; a first population of target nucleic acids,wherein the first population includes at least one target nucleic acidof a first type that binds to the first capture primers; a secondpopulation of target nucleic acids, wherein the second populationincludes at least one target nucleic acid of a second type that binds tothe second capture primers; b) forming a first amplified population ofnucleic acids by amplifying one or more target nucleic acids of thefirst population, wherein the first amplified population is attached toone or more beads of the first type; and c) forming a second amplifiedpopulation of nucleic acids by amplifying one or more target sequencesfrom the second population of target nucleic acids, wherein the secondamplified population is attached to one or more beads of the secondtype.
 2. The method of claim 1, wherein the forming the first amplifiedpopulation includes amplifying the first target sequence by PCR,isothermal, rolling circle, or emulsion based amplification.
 3. Themethod of claim 1, wherein the forming the second amplified populationincludes amplifying the second target sequence by PCR, isothermal,rolling circle, or emulsion based amplification.
 4. The method of claim1, wherein the first amplified population is covalently attached to oneor more beads of the first type.
 5. The method of claim 1, wherein thesecond amplified population is covalently attached to one or more beadsof the second type.
 6. The method of claim 1, wherein the singlereaction mixture includes at least two, three, four, five, six, seven,eight, nine, ten, or more types of beads, each type of bead including adifferent type of capture primer.
 7. The method of claim 1, furthercomprising sequencing one or more nucleic acids of the first amplifiedpopulation and one or more nucleic acids from the second amplifiedpopulation in parallel.
 8. The method of claim 7, wherein the sequencingincludes detection of one or more nucleotide incorporation byproducts.9. The method of claim 7, wherein the sequencing includes detectinghydrogen ions or pyrophosphate.
 10. The method of claim 1, wherein thefirst and second capture primers are present in about the sameconcentration.
 11. The method of claim 1, wherein the forming the firstamplified population includes hybridizing at least one target sequencefrom the first population to a first capture primer on the first type ofbeads; and the forming the second amplified population includeshybridizing at least one target sequence from the second population to asecond capture primer on the second type of beads.
 12. The method ofclaim 1, further including extending one or more first and secondcapture primers in a template-dependent fashion.
 13. The method of claim1, wherein the first amplified population is substantially monoclonal.14. The method of claim 1, wherein the second amplified population issubstantially monoclonal.
 15. The method of claim 1, wherein the firstamplified population, the second amplified population, or both, aresubstantially monoclonal.
 16. A method of monoclonally amplifying afirst target sequence from a first population of target nucleic acidsand a first target sequence from a second population of target nucleicacids comprising: (a) providing a single reaction mixture having aplurality of a first type of beads including first capture primers; aplurality of a second type of beads including second capture primers,wherein the first and second capture primers are different; a firstpopulation of target nucleic acids, wherein the first populationincludes at least one target nucleic acid that binds to a first fusionprimer; a first fusion primer that includes a portion complementary tothe first capture primers; a second population of target nucleic acids,wherein the second population includes at least one target nucleic acidthat binds to a second fusion primer; a second fusion primer thatincludes a portion complementary to the second capture primers; (b)forming a substantially monoclonal population of the first targetsequence from the first population of target nucleic acids on the firsttype of beads using the first fusion primer; and (c) forming asubstantially monoclonal population of the first target sequence fromthe second population of target nucleic acids on the second type ofbeads using the second fusion primer.
 17. A method for amplifying aplurality of different nucleic acid populations in a single reactionmixture, comprising: a) providing a single reaction mixture including afirst and a second population of nucleic acids, wherein the firstpopulation of nucleic acids contain a first primer binding sequence andthe second population of nucleic acids contain a second primer bindingsequence, and wherein the first and second primer binding sequences aredifferent; a first plurality of beads including a first capture sequencethat can bind to the first primer binding sequence; a second pluralityof beads including a second capture sequence that can bind to the secondprimer binding sequence, wherein the first and second capture sequencesare different; b) amplifying, within the single reaction mixture, one ormore nucleic acids from the first population to form a first amplifiedpopulation, and one or more nucleic acids from the second population toform a second amplified population, wherein the first amplifiedpopulation is bound to one or more beads of the first type and thesecond amplified population is bound to one or more beads of the secondtype.
 18. The method of claim 17, wherein the first amplifiedpopulation, the second amplified population, or both, are substantiallymonoclonal.
 19. The method of claim 17, wherein the amplifying in step(b) include PCR, isothermal, rolling circle, or emulsion basedamplification.
 20. The method of claim 19, wherein the emulsion basedamplification includes water-in-oil emulsions.
 21. The method of claim17, wherein the first amplified population is covalently bound to one ormore beads of the first type.
 22. The method of claim 17, wherein thesecond amplified population is bound to one or more beads of the secondtype.
 23. The method of claim 17, wherein the single reaction mixturefurther includes at least two, three, four, five, six, seven, eight,nine, ten, or more types of beads, each type of bead including adifferent type of primer binding sequence.
 24. The method of claim 17,further comprising sequencing one or more nucleic acids of the firstamplified population and one or more nucleic acids from the secondamplified population in parallel.
 25. The method of claim 24, whereinthe sequencing includes detection of one or more nucleotideincorporation byproducts.
 26. The method of claim 24, wherein thesequencing includes detecting hydrogen ions or pyrophosphate.
 27. Themethod of claim 17, wherein the first and second primer bindingsequences are present in about the same concentration.
 28. The method ofclaim 17, wherein the amplifying in step (b) includes hybridizing atleast one target sequence from the first population to a first primerbinding sequence on the first type of beads; and the forming the secondamplified population includes hybridizing at least one target sequencefrom the second population to a second primer binding sequence on thesecond type of beads.
 29. The method of claim 17, further includingextending one or more first and second primer binding sequence in atemplate-dependent fashion.
 30. The method of claim 17, wherein thefirst amplified population is substantially monoclonal.
 31. The methodof claim 17, wherein the second amplified population is substantiallymonoclonal.
 32. The method of claim 17, wherein the first amplifiedpopulation, the second amplified population, or both, are substantiallymonoclonal.